Computer-assisted methods and apparatus for identification and characterization of biomolecules in a biological sample

ABSTRACT

The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional of U.S. patent application Ser.No. 09/642,247, which is a Divisional of U.S. patent application Ser.No. 09/642,246, both filed Aug. 17, 2000, and both of which areDivisionals of U.S. patent application Ser. No. 09/339,164, which is aDivisional of U.S. patent application Ser. No. 09/339,165, now U.S. Pat.No. 6,136,173, issued Oct. 24, 2000, which is a Divisional of U.S.patent application Ser. No. 09/339,177, all of which were filed Jun. 24,1999, and all of which are Divisionals of U.S. patent application Ser.No. 08/881,761, filed Jun. 24, 1997, now U.S. Pat. No. 5,993,627, issuedNov. 30, 1999.

BACKGROUND OF THE INVENTION

[0002] The invention relates to the field of electrophoretic separationsof macromolecules and in particular, to the automation oftwo-dimensional electrophoretic separations used in the analysis ofproteins. Such two-dimensional procedures typically involve sequentialseparations by isoelectric focusing (IEF) and SDS slab gelelectrophoresis, and an automated 2-D method thus involves manufactureand use of gel media for both isoelectric focusing and SDSelectrophoresis, together with means for protein detection andquantitation. Two-dimensional electrophoresis technology forms the basisof the expanding field of proteomics, and hence automation of theprocedure is a critical requirement for scale-up of efforts to buildproteome databases comprising all the proteins of complex organisms suchas man. To date, no successful automation efforts have been reported,despite the use of bench-scale 2-D electrophoresis in more than 5,000scientific publications.

[0003] The publications and other materials used herein to illuminatethe background of the invention and in particular, cases to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience are referenced in the following text andrespectively grouped in the appended List of References. Elements of theinvention are disclosed in our Disclosure Documents 393753, 393754 and412899.

[0004] Isoelectric Focusing (IEF)

[0005] A protein is a macromolecule composed of a chain of amino acids.Of the 20 amino acids found in typical proteins, four (aspartic andglutamic acids, cysteine and tyrosine) carry a negative charge and three(lysine, arginine and histidine) a positive charge, in some pH range. Aspecific protein, defined by its specific sequence of amino acids, isthus likely to incorporate a number of charged groups along its length.The magnitude of the charge contributed by each amino acid is governedby the prevailing pH of the surrounding solution, and can vary from aminimum of 0 to a maximum of 1 charge (positive or negative depending onthe amino acid), according to a titration curve relating charge and pHaccording to the pK of the amino acid in question. Under denaturingconditions in which all of the amino acids are exposed, the total chargeof the protein molecule is given approximately by the sum of the chargesof its component amino acids, all at the prevailing solution pH.

[0006] Two proteins having different ratios of charged, or titrating,amino acids can be separated by virtue of their different net charges atsome pH. Under the influence of an applied electric field, a more highlycharged protein will move faster than a less highly charged protein ofsimilar size and shape. If the proteins are made to move from a samplezone through a non-convecting medium (typically a gel such aspolyacrylamide), an electrophoretic separation will result.

[0007] If, in the course of migrating under an applied electric field, aprotein enters a region whose pH has that value at which the protein'snet charge is zero (the isoelectric pH), it will cease to migraterelative to the medium. Further, if the migration occurs through amonotonic pH gradient, the protein will “focus” at this isoelectric pHvalue. If it moves toward more acidic pH values, the protein will becomemore positively charged, and a properly-oriented electric field willpropel the protein back towards the isoelectric point. Likewise, if theprotein moves towards more basic pH values, it will become morenegatively charged, and the same field will push it back toward theisoelectric point. This separation process, called isoelectric focusing,can resolve two proteins differing by less than a single charged aminoacid among hundreds in the respective sequences.

[0008] A key requirement for an isoelectric focusing procedure is theformation of an appropriate spatial pH gradient. This can be achievedeither dynamically, by including a heterogeneous mixture of chargedmolecules (ampholytes) into an initially homogeneous separation medium,or statically, by incorporating a spatial gradient of titrating groupsinto the gel matrix through which the migration will occur. The formerrepresents classical ampholyte-based isoelectric focusing, and thelatter the more recently developed immobilized pH gradient (IPG)isoelectric focusing technique. The IPG approach has the advantage thatthe pH gradient is fixed in the gel, while the ampholyte-based approachis susceptible to positional drift as the ampholyte molecules move inthe applied electric field. The best current methodology combines thetwo approaches to provide a system where the pH gradient is spatiallyfixed but small amounts of ampholytes are present to decrease theadsorption of proteins onto the charged gel matrix of the IPG.

[0009] It is current practice to create IPG gels in a thin planarconfiguration bonded to an inert substrate, typically a sheet of Mylarplastic which has been treated so as to chemically bond to an acrylamidegel (e.g., Gelbondg PAG film, FMC Corporation). The IPG gel is typicallyformed as a rectangular plate 0.5 mm thick, 10 to 30 cm long (in thedirection of separation) and about 10 cm wide. Multiple samples can beapplied to such a gel in parallel lanes, with the attendant problem ofdiffusion of proteins between lanes producing cross contamination. Inthe case where it is important that all applied protein in a given laneis recovered in that lane (as is typically the case in 2-Delectrophoresis), it has proven necessary to split the gel into narrowstrips (typically 3 mm wide), each of which can then be run as aseparate gel. Since the protein of a sample is then confined to thevolume of the gel represented by the single strip, it will all berecovered in that strip. Such strips (Immobiline DryStrips) are producedcommercially by Pharmacia Biotech.

[0010] While the narrow strip format solves the problem of containingsamples within a recoverable, non-cross-contaminating region, thereremain substantial problems associated with the introduction of sampleproteins into the gel. Since protein-containing samples are typicallyprepared in a liquid form, the proteins they contain must migrate, underthe influence of the electric field, from a liquid-holding region intothe IPG gel in order to undergo separation. This is typically achievedby lightly pressing an open-bottomed rectangular frame against theplanar gel surface so that the gel forms the bottom of an open-toppedbut otherwise liquid-tight vessel (the sample well). The sample is thendeposited in this well in contact with the gel surface forming thebottom of the well. Since all of the sample protein must pass through asmall area on the surface of the gel (the well bottom) in order to reachthe gel interior, the local concentration of protein at the entry pointcan become very high, leading to protein precipitation. The sample entryarea is typically smaller than the gel surface forming the well bottombecause the protein migrates into the gel under the influence of anelectric field which directs most of it to one edge of the well bottom,tending to produce protein precipitation. The major source ofprecipitation, however, is provided by the charged groups introducedinto the gel matrix to form the pH gradient in IPG gels: these groupscan interact with charges on the proteins (most of which are not attheir isoelectric points at the position of the application point andhence have non-zero net charges) to bind precipitates to the gel. It iscommon experience that separations of the same protein mixture on aseries of apparently identical IPG gels can yield very differentquantitative recoveries of different proteins at their respectiveisoelectric points, indicating that the precipitation phenomenon mayvary from gel to gel in unpredictable ways, thereby frustrating thegeneral use of IPG gels for quantitative protein separations.

[0011] Recently, methods have been introduced in which the IPG strip isre-swollen, from the dry state, in a solution containing sampleproteins, with the intention that the sample proteins completelypermeate the gel at the start of the run.

[0012] Isoelectric focusing separation of proteins in an immobilized pHgradient (IPG) is extensively described in the art. The concept of theIPG is disclosed in U.S. Pat. No. 4,130,470 and is further described innumerous publications. The IPG gel strips manufactured are generally ofsimple planar shape.

[0013] A series of disclosures have dealt with various configurations ofcavities (“sample wells”) used for the application ofmacromolecular-containing samples to the surfaces of gels, mostfrequently slab gels used for protein or nucleic acid separations. Ineach case, these sample wells were designed to concentratemacromolecules in the sample into a thin starting zone prior to theirmigration through the resolving gel. The following references describethe use of devices placed against a gel to form wells: U.S. Pat. No.5,304,292 describes the use of pieces of compressible gasket to formwell walls at the top of a slab where the ends of the pieces touch thetop edge of the slab. U.S. Pat. No. 5,164,065 describes a shark 's toothcomb used in combination with DNA sequencing gels.

[0014] Several references describe automated devices for creatinggradients of polymerizable monomers. Such systems have been used formaking porosity gradient gels used in molecular weight separations ofproteins. Altland, et al. (Altland, K. and Altland, A. Pouringreproducible gradients in gels under computer control, Clin. Chem. 30(12Pt 1): 2098-2103, 1984) shows the use of such systems for creating thegradients of titratable monomers used in the creation of IPG gels. U.S.Pat. No. 4,169,036 describes a system for loading slab-gel holders forelectrophoresis separation. U.S. Pat. No. 4,594,064 discloses anautomated apparatus for producing gradient gels. Hence, use of acomputer-controlled gradient maker in manufacturing IPG and other gelsis known in the art.

[0015] One alternative method of running IPG strips in an IsomorpHdevice is disclosed in Disclosure Document No. 342751 (Anderson, N. L.,entitled “Vertical Method for Running IPG Gel Strips”). The discloseddevice uses sample wells pressed against the gel surface, but otherwisecompletely closed, so that the assembly could be rotated into a verticalorientation, thus allowing closer packing of gels and a greater gelcapacity in a small instrument footprint. Additional methods aredisclosed in Disclosure Document No's 393753 (Anderson, N. L., Goodman,Jack, and Anderson, N. G., entitled “Gel Strips for Protein Separation”)and 412899 (Anderson, N. L., Goodman, Jack, and Anderson, N. G.,entitled “Automated System for Two-Dimensional Electrophoresis ”).

[0016] Systems for making non-planar slab gels are also known in the artand are disclosed in the following references: U.S. Pat. No. 5,074,981discloses a substitute for submarine gels using an agarose block that isthicker at the ends and hangs into buffer reservoirs. U.S. Pat. No.5,275,710 discloses lane-shaped gels formed in a plate and gel-filledholes extending down from the plate into buffer reservoirs. These gelsystems, however, do not provide a gel which can be given across-section that is optimal for producing high-resolution proteinseparation. Furthermore, these systems cannot incorporate varyingcross-sections along the length of a gel as required.

[0017] SDS Slab Gel Electrophoresis

[0018] Charged detergents such as sodium dodecyl sulfate (SDS) can bindstrongly to protein molecules and “unfold” them into semi-rigid rodswhose lengths are proportional to the length of the polypeptide chain,and hence approximately proportional to molecular weight. A proteincomplexed with such a detergent is itself highly charged (because of thecharges of the bound detergent molecules), and this charge causes theprotein-detergent complex to move in an applied electric field.Furthermore, the total charge also is approximately proportional tomolecular weight (since the detergent's charge vastly exceeds theprotein's own intrinsic charge), and hence the charge per unit length ofa protein-SDS complex is essentially independent of molecular weight.This feature gives protein-SDS complexes essentially equalelectrophoretic mobility in a non-restrictive medium. If the migrationoccurs in a sieving medium, such as a polyacrylamide gel, however, large(long) molecules will be retarded compared to small (short) molecules,and a separation based approximately on molecular weight will beachieved. This is the principle of SDS electrophoresis as appliedcommonly to the analytical separation of proteins.

[0019] An important application of SDS electrophoresis involves the useof a slab-shaped electrophoresis gel as the second dimension of atwo-dimensional procedure. The gel strip or cylinder in which theprotein sample has been resolved by isoelectric focusing is placed alongthe slab gel edge and the molecules it contains are separated in theslab, perpendicular to the prior separation, to yield a two-dimensional(2-D) separation. Fortunately, the two parameters on which this 2-Dseparation is based, namely isoelectric point and mass, are almostcompletely unrelated. This means that the theoretical resolution of the2-D system is the product of the resolutions of each of the constituentmethods, which is in the range of 150 molecular species for both IEF andSDS electrophoresis. This gives a theoretical resolution for thecomplete system of 22,500 proteins, which accounts for the intenseinterest in this method. In practice, as many as 5,000 proteins havebeen resolved experimentally. The present invention is aimed primarilyat the 2-D application, and includes means for automating the seconddimension SDS separation of a 2-D process to afford higher throughput,resolution and speed.

[0020] It is current practice to mold electrophoresis slab gels betweentwo flat glass plates, and then to load the sample and run the slab gelstill between the same glass plates. The gel is molded by introducing adissolved mixture of polymerizable monomers, catalyst and initiator intothe cavity defined by the plates and spacers or gaskets sealing threesides. Polymerization of the monomers then produces the desired gelmedia. This process is typically carried out in a laboratory setting, inwhich a single individual prepares, loads and runs the gel. A gasket orform comprising the bottom of the molding cavity is removed after gelpolymerization in order to allow current to pass through two oppositeedges of the gel slab: one of these edges represents the open (top)surface of the gel cavity, and the other is formed against its removablebottom. Typically, the gel is removed from the cassette defined by theglass plates after the electrophoresis separation has taken place, forthe purposes of staining, autoradiography, etc., required for detectionof resolved macromolecules such as proteins.

[0021] The concentrations of polyacrylamide gels used in electrophoresisare stated generally in terms of %T (the total percentage of acrylamidein the gel by weight) and %C (the proportion of the total acrylamidethat is accounted for by the crosslinker used).N,N′-methylenebisacrylamide (“bis”) is typically used as crosslinker.Typical gels used to resolve proteins range from 8% T to 24% T, a singlegel often incorporating a gradient in order to resolve a broad range ofprotein molecular masses.

[0022] In most conventional systems used for SDS electrophoresis, use ismade of the stacking phenomenon first employed in this context byLaemmli, U.K. (1970) Nature 227, 680. In a stacking system, anadditional gel phase of high porosity is interposed between theseparating gel and the sample. The two gels initially contain adifferent mobile ion from the ion source (typically a liquid bufferreservoir) above them: the gels contain chloride (a high mobility ion)and the buffer reservoir contains glycine (a lower mobility ion, whosemobility is pH dependent). All phases contain Tris as the low-mobility,pH determining other buffer component and positive counter-ion.Negatively charged protein-SDS complexes present in the sample areelectrophoresed first through the stacking gel at its pH ofapproximately 6.8, where the complexes have the same mobility as theboundary between the leading (Cl-) and trailing (glycine-) ions. Theproteins are thus stacked into a very thin zone “sandwiched” between Cl-and glycine-zones. As this stacking boundary reaches the top of theseparating gel the proteins become unstacked because, at the higherseparating gel pH (8.6), the protein-SDS complexes have a lowermobility. Thus, in the separating gel, the proteins fall behind thestacking front and are separated from one another according to size asthey migrate through the sieving environment of the lower porosity(higher %T acrylamide) separating gel. In this environment, proteins areresolved on the basis of mass.

[0023] Pre-made slab gels have been available commercially for manyyears (e.g., from Integrated Separation Systems). These gels areprepared in glass cassettes much as would be made in the user'slaboratory, and shipped from a factory to the user. More recently,methods have been devised for manufacture of both slab gels in plasticcassettes (thereby decreasing the weight and fragility of the cassettes)and slab gels bonded to a plastic backing (e.g., bonded to a Gelbond®Mylar® sheet or to a suitably derivatized glass plate). To date, allcommercially-prepared gels are either enclosed in a cassette or bondedto a plastic sheet on one surface (the other being covered by aremovable plastic membrane). Furthermore, all commercially-prepared gelshave a planar geometry.

[0024] Current practice in running slab gels involves one of twomethods. A gel in a cassette is typically mounted on a suitableelectrophoresis apparatus, so that one edge of the gel contacts a firstbuffer reservoir containing an electrode (typically a platinum wire) andthe opposite gel edge contacts a second reservoir with a secondelectrode, steps being taken so that the current passing between theelectrodes is confined to run mainly or exclusively through the gel.Such apparatus may be “vertical” in that the gel's upper edge is incontact with an upper buffer reservoir and the lower edge is in contactwith a lower reservoir, or the gel may be rotated 90° about an axisperpendicular to its plane, so that the gel runs horizontally between aleft and right buffer reservoir, as is disclosed in U.S. Pat. No.4,088,561 (e.g., “DALT” electrophoresis tank). Various configurationshave been devised in order to make these connections electrically, andto simultaneously prevent liquid leakage from one reservoir to the other(around the gel).

[0025] When used as part of a typical 2-D procedure, an IEF gel isapplied along one exposed edge of such a slab gel and the proteins itcontains migrate into the gel under the influence of an applied electricfield. The IEF gel may be equilibrated with solutions containing SDS,buffer and thiol reducing agents prior to placement on the SDS gel, inorder to ensure that the proteins the IEF gel contains are prepared tobegin migrating under optimal conditions, or else this equilibration maybe performed in situ by surrounding the gel with a solution or gelcontaining these components after it has been placed in position alongthe slab's edge.

[0026] A slab gel affixed to a Gelbond® sheet is typically run in ahorizontal position, lying flat on a horizontal cooling plate with theGelbond® sheet down and the unbonded surface up. Electrode wickscommunicating with liquid buffer reservoirs, or bars ofbuffer-containing gel, are placed on opposite edges of the slab to makeelectrical connections for the run, and samples are generally appliedonto the top surface of the slab (as in the instructions for thePharmacia ExcelGels).

[0027] It is current practice to detect proteins in 2-D gels either bystaining the gels or by exposing the gels to a radiosensitive film orplate (in the case of radioactively labeled proteins). Staining methodsinclude dye-binding (e.g., Coomassie Brilliant Blue), silver stains (inwhich silver grains are formed in protein-containing zones), negativestains in which, for example, SDS is precipitated by Zn ions in regionswhere protein is absent, or the proteins may be fluorescently labeled.In each case, images of separated protein spot patterns can be acquiredby scanners, and this data reduced to provide positional andquantitative information on sample protein composition through theaction of suitable computer software.

[0028] Additional methods are disclosed in Disclosure Document No's.393754 (Anderson, N. L., Goodman, Jack, and Anderson, N. G., entitled“Apparatus and Methods for Casting and Running Electrophoresis SlabGels”) and 412899 (Anderson, N. L., Goodman, Jack, and Anderson, N. G.,entitled “Automated System for Two-Dimensional Electrophoresis”).

[0029] Sample Preparation

[0030] Protein samples to be analyzed using 2-D electrophoresis aretypically solubilized in an aqueous, denaturing solution such as 9Murea, 2% NP-40 (a non-ionic detergent), 2% of a pH 8-10.5 ampholytemixture and 1% dithiothreitol (DTT). The urea and NP40 serve todissociate complexes of proteins with other proteins and with DNA, RNA,etc. The ampholyte mixture serves to establish a high pH (˜9) outsidethe range where most proteolytic enzymes are active, thus preventingmodification of the sample proteins by such enzymes in the sample, andalso complexes with DNA present in the nuclei of sample cells, allowingDNA-binding proteins to be released while preventing the DNA fromswelling into a viscous gel that interferes with IEF separation. Thepurpose of the DTT is to reduce disulfide bonds present in the sampleproteins, thus allowing them to be unfolded and assume an open structureoptimal for separation by denaturing IEF. Samples of tissues, forexample, are solubilized by rapid homogenization in the solubilizingsolution, after which the sample is centrifuged to pellet insolublematerial and DNA, and the supernatant collected for application to theIEF gel.

[0031] Because of the likelihood that protein cysteine residues willbecome oxidized to cysteic acid or recombine and thus stabilizerefolded, not fully denatured protein structures during the run, it isdesirable to chemically derivatize the cysteines before analysis. Thisis typically accomplished by alkylation to yield a less reactivecysteine derivative.

[0032] Use of2-D Electrophoresis

[0033] Two-dimensional electrophoresis is widely used to separate fromhundreds to thousands of proteins in a single analysis, in order tovisualize and quantitate the protein composition of biological samplessuch as blood plasma, tissues, cultured cells, etc. The technique wasintroduced in 1975 by O'Farrell, and has been used since then in variousforms in many laboratories.

[0034] The gel systems known in the art or referenced above, however, donot provide an integrated, fully automated, high-throughput system fortwo-dimensional electrophoresis of proteins. Moreover, current IPG andslab gel systems are not fully automated, wherein all operationsincluding gel casting, processing, sample loading, running and finaldisposition are carried out by an integrated, fully automated system.Current gel systems cannot be fully controlled by a computer and cannotsystematically vary gel, process, sample load and run parameters,provide positive sample identification, and cannot collect process datawith the object-of optimizing the reproducibility and resolution of theprotein separations.

OBJECT OF THE INVENTION

[0035] It is an object of the present invention to provide means forfully automated, high throughput two-dimensional electrophoresis ofproteins.

[0036] It is a further object of the present invention to provide ameans of alkylating protein sulfhydryl groups in an automated manner.

[0037] It is a further object of the present invention to provide an IPGgel system optimized for use in a two-dimensional gel system wherein alloperations including gel casting, processing, sample loading, runningand final disposition (either by staining for protein detection orapplication to a second dimension slab gel for use in a two-dimensionalprotein separation) are carried out by an automated system.

[0038] It is a further object of the present invention to provide an IPGgel which is not restricted to a planar geometry, but which instead canbe given any cross-section judged optimal for producing ahigh-resolution protein separation, and can incorporate varyingcross-sections along its length as required.

[0039] It is a further object of the present invention to provide an IPGgel strip system that can be fully controlled by a computer, therebyaffording the opportunity to systematically vary gel, process, sampleload and run parameters and collect process data with the object ofoptimizing the reproducibility and resolution of the separation.

[0040] It is a further object of the present invention to provide asystem for SDS slab gel electrophoresis offering facile automation (theslab gels should be easily handled in a robotic manner during casting,loading and running).

[0041] It is a further object of the present invention to provideaccurate placement of the sample with respect to the plane of the slabgel, so as to avoid migration of sample macromolecules in a distributionthat is asymmetric with respect to the plane of the slab gel, i.e.,along one surface.

[0042] It is a further object of the present invention to provideeffective and even cooling of the slab gel surface so that voltage (andhence heat generated) can be increased, with attendant improvements ingel resolution (due to shorted run times, and consequently decreaseddiffusion time).

[0043] It is a further object of the invention to provide facileautomation of slab gel staining and scanning.

[0044] It is a further object of the invention to provide automatedmeans for the recovery of selected protein spots or gel zones for thepurpose of protein identification and characterization by means such asmicrochemical sequencing or mass spectrometry.

SUMMARY OF THE INVENTION

[0045] The present invention provides an integrated, fully automated,high-throughput system for two-dimensional electrophoresis comprised ofgel-making machines, gel processing machines, gel compositions andgeometries, gel handling systems, sample preparation systems, softwareand methods. The system is capable of continuous operation athigh-throughput, to allow construction of large quantitative data sets.

[0046] Sample Preparation

[0047] Automated means are provided for treatment of protein-containingsamples to effect the reduction and alkylation of cysteine sulfhydrylgroups contained therein, with the object of preventing protein loss inthe 2-D process through protein aggregation or refolding associated withsulfhydryl re-oxidation during the run.

[0048] IEF

[0049] IPG gels are cast in a computer-controlled mold system capable ofrepeatedly casting a gel on a film support, advancing the support,cutting off the strip of support carrying the fresh gel, and presentingthe strip to a robotic arm. The robotic arm subsequently carries the IPGstrip and inserts it in a sequence of processing stations that implementsteps required to prepare the IPG and use it, including washing, drying,rehydration, sample loading, and subjection to high voltage.

[0050] The approach used in casting the IPG gel allows the shape of thegel to depart from conventional flat planar strip geometry. The methodof sample loading allows the sample to be applied over a large area ofthe gel. Such a gel format can provide an improved two-stage separationsystem: a first stage in which the proteins are separated in aminimally-restrictive, ideally fluid medium by isoelectric focusing in achannel or surface layer containing conventional ampholytes butsurrounded by an IPG gel that establishes the pH gradient, andcontinuing on to a second stage in which the proteins are imbibed by thesurrounding IPG gel at, or near their isoelectric points and maintainedin stable, focused positions until the end of the run.

[0051] SDS Slabs

[0052] SDS slab gels used for the second dimension separation are formedin an automated mold which plays the role of the gel-forming cassette ofa conventional system. By using an approach analogous to injectionmolding, the gel is no longer required to assume a homogeneous planarconfiguration. In effect, a three-phase gel may be constructed, havingregions corresponding to the separating gel, stacking gel and upperbuffer reservoirs of a conventional slab gel system. Polymerizable gelsolutions can be fed to the mold by one or more computer-controlledpumping devices, thus facilitating the creation of multiple zones of gelhaving different electrochemical properties. An upper electrode in theform of a rigid bar is polymerized into one region of the slab gel,allowing it to be manipulated and transported “bare” (i.e., without anysurface protection or coating) by a second robotic arm (i.e., nocassette).

[0053] A slot or other means is provided for introducing a sample(usually in the form of a first dimension gel rod or strip) into or ontothe slab. The slab is “run” (voltage applied) while it is hanging in abath of cooled, circulating insulating liquid, such as silicone oil. Theoil prevents evaporation of water from the planar gel surfaces as thegel runs (a function typically performed by the glass plates of aconventional gel cassette) and prevents joule heat caused by theelectrophoresis current from raising the temperature of the gelappreciably. The gel contacts a layer of aqueous solvent underlying theoil, serving as a lower buffer (with suitable electrodes). The lowdensity of the oil keeps it above and unmixed with the lower aqueousbuffer.

[0054] After the run, the slab gel is carried by the second robotic armto a succession of tanks containing a series of solutions needed toeffect staining of the protein spots or bands on the gel. Because ofdifferences in the physical densities of these solutions, the stainingcan make use of the fact that, as solutes are exchanged between thehanging gel slab and the solution, a lamina forms at the surfaces of theslab gel that has a density different from that of the bulk solvent.Because of this difference, the fluid in this lamina either rises orfalls as a curtain along the slab surface, and is replaced by freshsolvent. Hence, depleted solution accumulates at either the top orbottom of the tank, where it can be removed and replaced with freshsolution. After staining, the gel can be transported by the robotic armto a scanner where it can be digitized for computer analysis.

[0055] Software

[0056] The entire process can be controlled by a computer runningsoftware that both drives the creation and processing of each gel andcollects process data from sensors placed at strategic points in theproduction line so as to allow quality control and optimization. Ascheduling algorithm is implemented in software so that each sample canbe run with different gel parameters, if desired, while ensuring thatthe manifold actions required to process one sample do not interferewith actions required to process other gels in the system (e.g., so thatthe arm used to transport IPG gels between processing stations is notrequired to be in two places at once).

BRIEF DESCRIPTION OF THE DRAWINGS

[0057]FIG. 1 is a schematic diagram of the entire automated 2-Delectrophoresis process.

[0058]FIGS. 2A through 2H illustrate sample preparation using a sizeexclusion column.

[0059]FIG. 3 illustrates an IPG gradient maker and mold system.

[0060]FIGS. 4A and 4B are schematic cross-sections through an IPG moldsystem.

[0061]FIGS. 5A through 5F shows a series of six alternativecross-sections for IPG gels formed by various mold activities.

[0062]FIG. 6 is a schematic view of an IPG strip in a horizontalposition with the gel-side on top of a base plate in position for sampleloading.

[0063]FIG. 7 is a side view of an IPG carrier arm and an IPG slot run.

[0064]FIGS. 8A through 8E illustrate the sequence of actions of a slabgel mold during casting operation.

[0065]FIGS. 9A through 9K illustrate alternative forms of slab gels.

[0066]FIG. 10 is an end view of slab gel run tanks.

[0067]FIG. 111A is an end view of slab gel staining tanks with the slabgel carrier arm,

[0068]FIG. 11B is an end view of the a slab gel in the holder, and

[0069]FIG. 11C is an end view of a slab gel held by a clamp.

[0070]FIG. 12 illustrates the placement of a slab gel on a scanningplatform by a slab carrier arm and configuration of fluorescenceillumination.

[0071]FIG. 13 illustrates the sequence of actions of a spot excisionpunch.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0072] The preferred embodiment of the automated system for 2-Delectrophoresis described is a continuously-operating production line,making each gel (both IEF and SDS) just as needed, and capable ofundertaking all steps of the process (FIG. 1)—from loading of sampleonto the first dimension gel to final entry of protein quantitation datainto a computer database.

[0073] Sample Preparation

[0074] In order that proteins retain constant chemical properties duringthe process of separation by IPG-IEF and SDS electrophoresis, it isimportant that the sulfhydryl (SH) groups of the cysteine residues thatthey contain not be allowed to reform disulfide bridges or becomeoxidized to cysteic acid during the separation process. In the preferredembodiment, the protein cysteine residues are permanently renderedstable by alkylation with iodoacetamide or one of its uncharged orzwitterionic derivatives (such as S+2-amino-5-iodoacetamido-pentanoicacid), which introduces a very hydrophilic group at every cysteineposition but does not change the protein's net charge or apparentisoelectric point and has a negligible effect on protein mass. Thisderivatization is implemented in an automated fashion using a sizeexclusion gel filtration column to exchange the proteins out of theinitial sample solubilization solution, through a reagent zonecontaining alkylating reagent, and finally into a medium suitable forapplication to an IPG gel. The size exclusion media is chosen so as toexclude proteins but not low molecular weight solvents (e.g.,polyacrylamide beads such as BioRad P-6 BioGel). In practice, a samplecontaining a sulfhydryl reducing reagent such as DTT is removed from avial selected by a conventional autosampler such as is used in highperformance liquid chromatography (HPLC), directed by a valve at thehead of the column onto a column which has been pre-equilibrated withthe final sample solvent and a zone (immediately preceding the sample)containing alkylating reagent in sufficient excess to ensure rapidreaction with protein cysteines. Once the sample zone is loaded, thevalve switches to deliver a stream of final sample solvent that propelsall the zones down the column and prepares the column for the succeedingcycle. As the initial sample zone moves down the column the proteinmolecules, because of their greater size, fail to penetrate into theparticles of the column packing and hence move forward at a greaterspeed than that of the bulk solvent, which freely exchanges into thevolume of the porous particles. This principle of separation is wellknown in the art. The proteins thus move into the zone of alkylatingreactant, react there, and finally move even farther forward into thepreceding zone of final sample solvent. This procedure thus ensuresalkylation of protein sulfhydryls and removal of any low molecularweight contaminants as well. The sample is then ready for application toan IPG gel.

[0075]FIG. 2 illustrates a sample preparation apparatus which uses asize exclusion column. FIG. 2A depicts the arrangement of the componentsof the sample preparation apparatus. A size exclusion column 1 isconnected to one of a series of input liquid streams 2, 3 and 4 by amulti-position switching valve 5, with liquid flow into the columndriven by pump 6. Initially column 1 is equilibrated with liquid 4.Input 2 delivers the crude sample from a conventional autosampler orother device. Input 3 delivers a stream of reagent required to effect achemical treatment of the sample proteins (typically a sulfhydrylalkylating reagent), and input 4 delivers a stream of column eluent (thesolvent in which the sample proteins will ultimately emerge). Liquidemerging from the size exclusion column flows through a UV absorbance orother column monitor flow cell 7 and thereby to a multiport valve 8 thatdirects the eluent either to waste 9 or to a sample collection vessel10.

[0076]FIGS. 2B through 2H depict steps in the operation of the column toeffect sample protein derivatization. In FIG. 2B, the column 1 isequilibrated with eluent through connection of its input to eluentreservoir 4 by input valve 5, and its output to waste 9 by output valve8. Pump 6 and UV monitor 7 are not shown for clarity: the pump isassumed to remain on during the sequence of operations, deliveringliquid continuously through the column. In FIG. 2C, a zone of alkylationreagent 11 is introduced onto the column by switching the input valve 5to draw solvent from the alkylation reagent reservoir 3. In FIG. 2D, azone of sample is introduced after the alkylating reagent zone, saidsample zone comprising a solvent phase 12 and a protein solute phase 13.In FIG. 2E, input to the column once again switches to eluent, pushingthe sample and alkylation zones down the column.

[0077] As the sample solvent zone moves down the column, the proteins itinitially contained are excluded from the matrix of the size exclusioncolumn and hence advance into the alkylation zone (a well known featureof such columns when used in desalting applications). During thisperiod, the proteins are exposed to the alkylating reagents and theircomponent sulfhydryl groups are alkylated to prevent re-folding of theproteins in subsequent stages of the 2D electrophoresis process. In FIG.2F, the proteins in solute phase continue to advance down the columnfaster than the proteins in solvent phase, and enter the leading regioncomprised of the first applied eluent phase. In FIG. 2G, the alkylatedproteins are collected by switching the output collection valve 8 to thesample collection position. In FIG. 2H, continuing flow of eluent intothe column forces the alkylation and initial sample solvent phases outof the column in preparation for the column's regeneration and re-use.

[0078] In an alternative embodiment, alkylation is performed with anegatively charged reagent such as iodoacetic acid, thereby substitutinga negative charge at every alkylated protein sulfhydryl. When thisreaction is accomplished stoichiometrically, very basic proteinscontaining cysteine residues are shifted towards more neutralisoelectric points, thereby facilitating their detection on IEF gels.

[0079] IPG

[0080] The first operation of the 2D gel procedure is creation of anisoelectric focusing gel to effect the first dimension separation. Sucha separation is most effectively carried out in an immobilized pHgradient (IPG) gel, in which a gradient of polymerizable monomers isgelled to form a fixed spatial pH gradient.

[0081] Gradient

[0082] The compositional gradient required to form the desired pHgradient IPG gel can be produced by a system of four computer-controlledmotorized syringes delivering, respectively, heavy gel monomercomposition formulated to yield a basic pH, light gel monomercomposition formulated to yield an acidic pH, a polymerization initiatorsuch as ammonium persulfate, and a polymerization catalyst such asTEMED. A computer program constructed, for example, in the LabVIEWlanguage, is used in conjunction with a computer and stepper motorcontrol card (for example, a Compumotor AT6400 card) to produce avarying ratio between the speed of delivery of heavy and lightcomponents, while maintaining a continuous delivery of initiator andcatalyst required for polymerization. Each of the four syringes isconnected to a separate computer-controlled valve (e.g., a 6-port highpressure liquid chromatography valve in which each of two rotationalpositions connects a fixed input with one of two lines and a fixedoutput with one of two other lines) that allows connection of thesyringe either to an external reservoir, or to the delivery tubingsystem. When the syringe is connected to the reservoir for refilling,the delivery system is connected to a source of pressurized flushsolvent (typically water) that displaces polymerizable monomer solutionsfrom the delivery tubes to prevent blockage. In the delivery tubingsystem, the four component flows emerging from the four valves arecombined by appropriate tubing junctions to yield one mixed fluid streamrouted into the gradient delivery tube in the mold.

[0083] An additional fifth syringe may be added to supply a thirdpolymerizable monomer solution of density and pH intermediate betweenthe light and heavy monomer solutions, for the purpose of creating verywide pH gradients as a sequence of two two-component gradients (i.e.,A->B followed by B->C).

[0084]FIG. 3 schematically depicts the components of an IPG castingsystem. A vertically-oriented mold cavity is formed of a front mold half14 and a back surface comprised of activated Gelbond® sheet 15. At eachcasting cycle, fresh Gelbond® is delivered to the mold from a roll 16through motorized transport rollers 17. A small diameter rigid deliverytube 18 extends into the mold from the top and may be raised out of themold by linear motion 19. A flexible tube 20 delivers a polymerizablecomposition to the delivery tube from a gradient maker having fivecomputer controlled syringes. Each syringe 21 is connected to the outputmanifold 22 through a 6-port valve 23 allowing the syringe to beconnected either to a liquid reservoir (e.g., liquid reservoir 24) forrefilling or to the output manifold. These syringes deliver one of threeacrylamide monomer solutions (24, 25, 26) ammonium persulfate 27 andTEMED 28. Valves attached to syringes drawing from reservoirs 24, 26, 27and 28 are shown in delivery position, while the valve attached to thesyringe drawing from reservoir 25 is shown in refill position.

[0085] Each syringe is driven by a motor 29 rotating a lead screw 30that generates linear motion of a block 31 attached to the syringe'splunger 32. During the refilling of syringe 21 from its associatedreservoir 24, the associated 6-port valve 23 connects the outputmanifold 22 to a pressurized source of non-polymerizable solvent 33(e.g., water), to purge the manifold and delivery tubes of polymerizablemedia (this configuration shown for the middle syringe connected toreservoir 25). After delivering a gradient of polymerizable monomers tothe mold, the delivery tube 18 is raised by delivery tube motion 19 sothat its open end lies in a block 34 through which air is sucked at highvelocity by an air pump, from input 35 to output 36. A second linearmotion 37 carries a long straight pin 38 which can be inserted into themold along its axis or raised out of it.

[0086] The resulting compositional gradient must be delivered into asuitable mold such that a spatial gradient is maintained duringgelation. In order to achieve this, the delivery tube delivering gelcomposition to the mold is arranged on a vertical linear transportcapable of inserting the open end of the delivery-tube to the bottom ofthe vertical mold cavity, and raising it slowly as the gradient isdispensed so as to deposit successive elements of the gradient above oneanother (at the rising meniscus of the liquid in the mold). When thegradient is thus completed, the delivery tube is raised fully out of themold and into a suction block 34 mounted just above the gel mold. Inthis position, liquid emerging from the delivery-tube is sucked into aperpendicular waste tube by the action of a vacuum, thereby providing awaste path for flush solvents directed through the delivery-tube betweengradient dispensing operations in order to prevent blockage of the tubeby any remaining polymerizable components.

[0087] Suitable compositions for the four components combined to make anIPG are as follows. Solutions of polymerization catalyst and initiator(assuming that each comprises 10% of the total volume dispensed) are,respectively, 1.2% tetramethylethylenediamine (TEMED) and 1.2% ammoniumpersulfate (AP), both in water. The two solutions of polymerizablemonomers (whose proportions in the output stream vary to yield agradient of titratable monomers and physical density) may be made up asshown in the following Table to achieve a gradient over a range of pH 4to 9. The titratable monomers used are immobilines® manufactured byPharmacia Biotech. Glycerol and deuterium oxide (heavy water) are usedto increase the density of one of the solutions, thus helping tostabilize the gradient formed in the mold through the interaction of theresulting density gradient and the earth's gravity. TABLE 1 Heavy Light(pH 4) (pH 9) Immobiline pK 3.6 2,762 491 microliters Immobiline pK 4.6785 1,414 microliters Immobiline pK 6.2 773 1,200 microliters ImmobilinepK 7 75 988 microliters Immobiline pK 8.5 834 236 microliters ImmobilinepK 9.3 738 2,209 microliters 31.8% T, 5.6% C Acrylamide/bis in 0 9.83 mlH₂O 31.8% T, 5.6% C Acrylamide/bis in 9.83 0 ml D₂O Glycerol 6.25 0 mlD₂O (Heavy Water) 19.79 0 ml Water 0 25.46 ml 1 M Tris HCI, pH 7.0 8.178.17 ml Total 50.00 50.00 ml

[0088] Because the volume of the tubing connecting the gradient makerwith the mold is a significant fraction of the mold volume (even whennarrow-bore HPLC tubing and connectors of inside diameter 0.010″ areused), it is necessary to take account of this volume when dispensing agradient. Hence, the procedure adopted and implemented in the controlsoftware consists of five stages: 1) delivery of the first segment ofthe desired gradient, equal in volume to the volume of the deliverytube, for the purpose of replacing the flush solvent in the tube withpolymerizable monomer; 2) insertion of the delivery tube into the mold;3) delivery of the remainder of the gradient while the delivery tube israised (withdrawn from the mold) at a speed such that the deliveredgradient composition is emitted at the rising surface of the liquid inthe mold; 4) following the gradient by a volume equal to the deliverytube volume of the final “light” composition, for the purpose of forcingthe section of the gradient remaining in the delivery tube into the moldwhile the delivery tube continues to rise; and 5) removal of thedelivery tube from the mold to the upper vacuum flush position where,following switching of the four valves, flush liquid is forced throughthe delivery tube system to remove polymerizable material and to preparethe system for a subsequent gradient delivery.

[0089] Mold

[0090] In the preferred embodiment, the IPG is cast in a narrow verticalmold cavity formed by pressing a movable mold half against a sheet ofGelbond® PAG-activated plastic substrate which in turn is pressedagainst a fixed backing block whose temperature is controlled bycirculation of chilled or heated water through internal cavities. Thecavity in the movable mold half is surrounded on the sides and bottom byan O-ring groove with an O-ring to produce a liquid-tight seal againstthe Gelbond®. The Gelbond® substrate is made of Mylar® polyester plasticfilm treated in such a way as to produce on its active surface groups towhich an acrylamide gel can bond covalently, thus attaching the gel tothe Gelbond® substrate.

[0091] In the preferred embodiment, a longitudinal IPG gradient isformed in the cavity by dispensing a varying composition of gelablemonomers into the cavity through a small diameter delivery tube. Thisdelivery tube rises during the dispensing of the gradient, andconsequent filling of the mold, so that the open end of the tube fromwhich gelable monomer emerges is maintained at the rising level of thesurface of the liquid dispensed into the mold. In addition, the gradientof gelable monomers is contrived so as to incorporate a physical densitygradient that evolves from heavy to light during the dispensing of thegradient. Such a density gradient is produced by inclusion of a densesubstance such as glycerol or deuterium oxide in place of a portion ofthe water present in the “heavy” gradient component. A density gradientdispensed in the “heavy” to “light” sequence from a tube maintained atthe rising surface of liquid in the mold gives rise to a stablecomposition gradient in the mold which, when polymerized, yields an IPG.

[0092]FIG. 4 is a schematic cross-section of an IPG mold system viewedfrom above (i.e., looking down into the mold cavity 14 depicted in FIG.3). In FIG. 4A, the front IPG mold half 14 is pressed against theGelbond® sheet 15 by a pneumatic cylinder 39, whose pressure bears on afixed back plate 40. In this example, the IPG mold cavity is of asemi-circular cross section 41. Lateral leakage of polymerizablecomponents is prevented by linear O-rings 42. Mold temperature can becontrolled by circulation of hot or cooled liquid through internalchannels of the fixed back plate. Polymerizable components areintroduced into the mold through moving delivery tube 18. A central holemay be formed in the IPG gel by polymerization with a pin 38 in placeinside the mold cavity.

[0093] In FIG. 4B, following extraction of the delivery tube and pinfrom the mold, pneumatic cylinder 39 retracts the front IPG mold half14, and rollers 17 cause the Gelbond® support 15 to slide laterallyacross the face of fixed back plate 40, thereby ejecting the IPG gel 43on its Gelbond® substrate from the mold, in preparation for anothercycle. A rotary blade 44 cuts the Gelbond® by moving vertically alongthe mold, thereby releasing a strip of Gelbond® carrying thenewly-formed IPG gel. The gel produced has a longitudinal hole 45.

[0094] It is important to ensure that the gradient of the resulting gelreaches hydrostatic equilibrium (and hence proper gradient shape) beforepolymerization, and yet is fully polymerized (with completeincorporation of gelable monomers into the gel polymer matrix) beforeremoval from the mold. This result is achieved by increasing thetemperature in the mold after an initial gradient formation period:gelation proceeds much faster at higher temperature. In a typicalprotocol, the gel gradient is introduced at a temperature of 20° C. andafter a period of approximately 4 minutes, during which thepolymerizable monomers gel into a non-convecting state, the temperaturein the mold is increased to approximately 50° C. by circulation ofheated water through closed channels provided in the backing plate.After removal of the gel from the mold, the temperature is lowered to20° C. by switching the circulation system to a chilled water supply inpreparation for the next cycle.

[0095] Once the gel is polymerized, the mold is opened and the IPG gelis transported by manipulation of the Gelbond® support to which it hasbecome covalently attached during polymerization. The form of the gel isdetermined by the form of the mold in which it is cast, the simplestbeing a flat, rectangular strip on the surface of the Gelbond®.

[0096] In a further embodiment, the gradient stream of polymerizablemonomers is introduced into the mold cavity by means of a passage at thebottom of the cavity, in this case in the sequence light to heavy(opposite to the order when liquid is deposited at the rising surface ofthe liquid in the mold). A special valve is used to direct the flow ofpolymerizable liquid either into the mold or to waste, thereby allowingthe contents of the delivery tubing to be purged of polymerizablecomponents after casting of a gel.

[0097] Numerous alternative forms of the IPG gel can be produced. In onealternative embodiment, a pin is introduced into the mold before orduring gel polymerization and slowly withdrawn afterwards, leaving acentral hole down the length of the IPG gel. This can be accomplished bya procedure in which the pin is first rotated slowly, to reduce theadhesion of gel to the pin, and subsequently slowly withdrawn along itsaxis through the top of the mold. In another embodiment, a sampleintroduction channel or groove is formed at the exterior surface of theIPG gel by means of a suitably shaped ridge on the interior surface ofthe mold. The groove may be formed so as to be closed at its ends, thusforming a bounded depression, open only at the top. Provided that thegel is held horizontal during the run, i.e. with the groove in ahorizontal plane and with its opening directed upward or to the side,then sample liquid placed in this groove will remain held there bycapillary action, until imbibed by the gel or evaporated.

[0098]FIG. 5 shows a series of six alternative cross-sections for IPGgels formed by various mold cavities, after the strip has been cut fromthe Gelbond® roll. In FIG. 5A, a semi-circular gel 43 with longitudinalhole 45 has been formed on the Gelbond® strip 46 and subsequently filledwith sample. In FIG. 5B, a semi-circular cross-section gel has a surfacegroove 47 in which sample is held by capillary action, while in FIGS. 5Cand 5D, other cross-sections with broader, flatter surface grooves 47are shown, also holding sample by capillarity. In FIGS. 5E and 5F,triangular and rectangular cross-sections without surface grooves areshown. In each case shown, the Gelbond® backing material is wider thanthe gel itself, giving the strip greater stiffness and providing,particularly in FIGS. 5E and 5F, a further form of cavity in whichsample is held by capillarity: a groove created by the included anglebetween the side of the gel on one hand and the extended Gelbond®substrate on the other.

[0099] In practice, the gel mold can be formed from any of a range ofmaterials that do not inhibit polymerization of acrylamide, includingglass, alumina, machinable ceramic, Ultem®, polysulfone, polystyrene,polycarbonate, polyurethane, acrylic, polyethylene or the like. Forconvenience in machining, and to allow observation of the mold'scontents, a clear plastic such as polysulfone or acrylic is preferred.

[0100] Gelbond® TransVort

[0101] Gelbond® substrate is advanced to the mold on repeated cyclesfrom a large roll by feed rollers. After casting an IPG gel on the endof the Gelbond® (the IPG axis perpendicular to the length of theGelbond® and parallel to the roll's axis), the strip of Gelbond® onwhich the gel is formed is cut from the roll using any of a variety ofmechanical cutting mechanisms, including, for example, a rolling diskcutter of the type used to cut photographic paper, affixed to a verticalmotion device. The resulting Gelbond® strip with IPG gel attached maythen be grasped by any of a variety of mechanical or manual means forhandling in further processing steps. In the preferred embodiment, thestrip is 1.27 cm wide and approximately 65 cm long (the width of theGelbond® substrate when provided in roll form). The IPG gel is 2 mmwide, 0.75 mm thick and 57 cm long (leaving 1.5 cm of the Gelbond®uncovered on either end of the strip).

[0102] Barcode Labeler

[0103] Preprinted barcoded labels are mechanically applied to eachIPG-carrying strip on the side opposite the gel for identificationpurposes, although other labeling means known or available may be used.

[0104] Robotic Arm

[0105] A robotic arm system equipped with two pneumatically-activatedpincers grasps the strip by the two ends to transport it betweensubsequent processing stations. The IPG arm system moves horizontallyalong a track, vertically along a linear table mounted on the track, andcan rotate 90 degrees in order to pick up the IPG lying in a horizontalposition and carry it in a vertical orientation to subsequent stations.

[0106] Sequence of IPG Processing Events

[0107] To employ a gel made by the procedure described above as thefirst dimension separation of a 2-D electrophoresis procedure, asequence of processing operations, many of which have been welldescribed in the art, is used to render the gel ready for use in aprotein separation. These operations include removal of remainingunpolymerized monomers, initiator and catalyst by washing in deionizedwater; dehydration to remove incorporated water; and finally rehydrationin a solution appropriate as a medium for protein separation.Subsequently, a protein-containing sample is applied to the gel, and thegel is subjected to a voltage gradient in order to separate the proteinsalong the gel length.

[0108] In the preferred embodiment, the IPG gel on its Gelbond® strip isgripped at both ends by the aforementioned movable arm and placed in oneof a plurality of slots containing circulating purified water. Afterapproximately two hours, most soluble materials remaining in the gelhave diffused into the water and are thus removed from the gel.

[0109] The strip is then grasped again by the arm (which in the meantimemay have moved to other positions to carry out other functions) andmoved to a slot where it is subjected to a stream of air filtered so asto remove any contaminating particulate material (e.g., using aconventional HEPA filter). The gel is substantially dried inapproximately 30 minutes.

[0110] Next, the arm again grasps the strip and moves it to a slotfilled with rehydration solution, a medium typically consisting of 9mole/liter urea, 2% of a non-ionic detergent such as Nonidet P40 orCHAPS, and 2% wide range, commercially-available ampholytes (e.g., BDH3-10 ampholytes) in water. When samples are to be used whose protein SHgroups have not been alkylated, 1% dithiothreitol is included in therehydration solution as a sulfhydryl reducing agent. In a period ofapproximately two hours, the IPG gel is re-swollen in rehydrationsolution and ready to be used for protein separation. In order toprevent the formation of crystals due to evaporation at the surface ofthe rehydration solution bath, the rehydration solution is covered by alayer of light silicone oil, through which the IPG is inserted.

[0111] To carry out a protein separation, a volume of sample proteinmust be applied to the gel. In the preferred embodiment, sample proteinin a solubilization solution similar in composition to the rehydrationsolution is applied on the surface of the IPG gel along its length. Thisapplication is effected by placing the IPG on a base plate with the gelface up, and depositing a stream of sample liquid onto the IPG gelsurface from a needle held just above that surface, which is movedslowly along the length of the IPG as sample is pumped out. Theresulting thin layer of protein-containing liquid on the IPG gel surfaceremains in place during subsequent manipulations of the gel strip solong as the axis of the gel remains in a horizontal plane (as is thecase during movement using the arm system described). Means are providedfor moving the needle up and down (to allow collection of sample bypiercing the septum of a conventional septum-topped sample vial), andfor moving it along the length of the IPG and farther, to positionswhere a sample vial may be placed and where the needle may be washed.

[0112]FIG. 6 shows an apparatus for application of sample protein to anisoelectric focusing gel in accordance with the present invention. AnIPG strip 46 lies horizontally, gel-side up, on a base plate 48. A trailof sample liquid 49 is left on the surface of the IPG gel as needle 50discharges a steady stream of sample while moving along the IPG. Theneedle 50 is moved on carriage 51 through the action of lead screw 52driven by motor 53. Sample flow is controlled by syringe 54 whoseplunger 55 is moved by a block 56 which is in turn moved by a lead screw57 turned by motor 58. Flexible tube 59 connects the syringe and thedelivery needle. The sample is initially taken into the needle 50 andsyringe 54 by raising the needle on its vertical pneumatic motion 60,driving the needle 50 to the left, positioning it over sample vial 61,lowering the needle 50 to pierce the vial's septum top, withdrawing thesample through action of the syringe 54, raising the needle 50 again,moving into position over a gel strip, lowering the needle 50 andcommencing synchronous motion of the syringe 54 and the needle carriage51 to deposit the sample along the IPG surface. The needle 50 is washedbetween applications by positioning it over waste receptacle 62, whereits exterior surface is washed by a jet of water 63.

[0113] In another embodiment, where a central hole is produced in theIPG gel during casting, the sample can be injected or peristalticallydrawn into the channel prior to application of voltage along the gel.The sample liquid can be retained inside the channel by pinching theends of the gel to close the channel, by injection of gas bubbles, or byvarious other means, including placing a drop of gelling material atboth ends.

[0114] After sample loading, the gel strip is once again grasped by thearm and moved to one of a plurality of slots tilled with anon-conducting oil (such as silicone oil) and having slotted carbonelectrodes at either end positioned so as to contact the ends of the IPGgel. The oil may be circulated, cooled to ensure constant runningtemperature and sparged with a dry gas so as to eliminate oxygen anddissolved water. Since the resistance of the IPG gel rises during therun, slots maintained at a series of different voltages are provided,and the arm periodically moves the strip from one voltage to a highervoltage as the run progresses. In the preferred embodiment, a series of6 voltage stages are provided, namely 1, 2.5, 5, 10, 20 and 40kilovolts. The gel is maintained at each voltage for about 3 hours,except the last, where it rests until a second dimension slab gel isavailable. A total of 200,000 to 300,000 volt-hours may applied to eachgel.

[0115] Slots such as those used for washing and for subsequentprocessing and running steps generally have clips at either end intowhich the gel strip is inserted by the arm, using a downward motion.When the grasping pincers at the ends of the arm release the Gelbond®strip, these clips continue to hold the strip extended between them byfriction. In the preferred embodiment, these clips consist of a pair ofparallel pins touching one another and projecting upwards from the floorof the slot. The strip is jammed between these pins during insertioninto the slot, spreading them slightly and producing a friction fit. Allthe slots except the air dryer are contained at the sides and below toyield a liquid-tight vessel suitable for containing the liquid withwhich the IPG is to be treated at that stage. Slots used for applicationof high voltage also contain slotted carbon electrodes.

[0116]FIG. 7 shows a cross-section view of an IPG processing slot andthe arm used to transport IPG strips between slots. A Gelbond® strip 46carrying attached IPG gel 43 is held at its ends 64 and 65 by a distalarm 70 and a proximal arm 68, each carrying a gripper 66 actuated by apneumatic cylinder 67. Both arms are mounted on a horizontal bar 69. Oneof the arms, in this case the distal arm 70, is mounted to a carriage 71capable of moving along bar 69 under the control of belt 72, which inturn is moved by motor and pulley 73. Since the other arm 68 is fixed tothe horizontal bar 69, movement of arm 70 by the motor and pulley in anoutwards direction serves to stretch strip 46, keeping it taut (andtherefore straight) between grippers 66. A vertical motion 74 serves toraise and lower the entire arm and bar assembly, thus allowing insertionof IPG gels into, and removal of gels from, the slots. The verticalmotion is itself carried on motor-driven wheels 75 which engage a track76 to move the arm assembly to positions over a variety of slots.

[0117] Movement of the arm assembly downwards (by motion of verticalmotion 74) causes gel strip 46 and attached IPG gel 43 to be insertedinto a processing slot in plate 77. The strip is held at its endsbetween pairs of pins 78 projecting from the floor of the slot, and isinserted beneath the surface of liquid 79. This liquid can be circulatedover the IPG strip by introducing liquid through inlet 80 andsimultaneously withdrawing liquid through outlet 81. Excess liquid flowsover a dam 82 to exit via overflow 83. In slots devoted to the IEFprocess (where voltage is applied across the gel) the ends of the IPGgel 43 contact slotted electrodes 84, which are connected in turn toconducting pins 85 that penetrate the bottoms of the run slots in aliquid-tight manner, allowing electrical connection to a power supply onthe outside.

[0118] During the early stages of a separation run, under an appliedelectric field, proteins can migrate through the liquid phase of theapplied sample along a pH gradient initially formed by the action of theampholytes incorporated in the sample. Because the proteins areinitially migrating through liquid, without the retardation associatedwith migration through a gel matrix, they can approach their isoelectricpoints more rapidly than in a system where the entire migration path isthrough IPG gel. However, if proteins remained in this liquid phase atthe end of the run, they could be displaced from their isoelectricpositions by subsequent gel handling steps. Hence, conditions arecontrived so that, as the run progresses, sample-containing liquid isimbibed by the gel, progressively shrinking the channel so that at theend of the run the channel contains a negligible amount of liquid. Thisis achieved by allowing surface water to be slowly removed from theexterior surface of the gel during the run by, for example, immersion ofthe gel in circulated silicone oil that has been dehydrated by spargingwith a dry gas such as argon or nitrogen.

[0119] During gel dehydration, and consequent collapse of any liquidfilled central sample channel, proteins enter the gel at positions neartheir respective isoelectric points. Thus, a mixture of differentproteins will enter the gel at points distributed along the gel length,rather than at one site at the edge of a sample well, thereby avoidingthe precipitation often observed when a complex mixture of proteinsmigrates together into the gel through a small gel surface area. Excessliquid is removed through the exterior gel surface, either to a dry gasphase or to a water-extracting, non-aqueous, non-conducting liquid phasesuch as silicone oil.

[0120] SDS Electrophoresis

[0121] Slab Gel Casting

[0122] In the preferred embodiment, a gel is formed in acomputer-controlled mold system whose operation is showndiagrammatically (in cross-section) in FIG. 8. The mold is composed oftwo halves 86 and 87 which can be forced together to comprise aliquid-tight cavity open at the top, The form of the mold is such thatthe gel 89 formed therein has a large, thin planar region at the bottom(within which proteins will be separated: the “separating gel”) andabove the thin planar region a substantially wider region (the “topgel”) joined to the thin region by a joining region of graduallyincreasing width. The function of the top gel is to provide a bufferreservoir as a source of ions during the electrophoresis separation, anda mechanical support from which the separating gel hangs during the runand subsequent steps. The joining region joins the separating and topgel regions and provides a gradually narrowing cross-section adapted forthe focusing of protein zones using the stacking process disclosed inLaemmli (U.K., 1970, Nature 227, 680), in which the joining region iscomprised of a stacking gel. In the preferred embodiment, the separatingregion has a thickness of about 1 mm, the top region has a thickness ofabout 2 cm, and the joining region gives rise to a smooth fillet betweenthe separating and top gels. The vertical height of the separating gelis 30 cm and that of the top gel is 5 cm. All gel regions have the samewidth, namely 60 cm.

[0123] Mixtures of polymerizable gel monomers are introduced into theclosed mold by means of three tubes 88, 90 and 95 which can be made toextend down into the mold cavity from above. The first delivery tube 88can be caused to extend to the bottom of the mold and is used tointroduce a liquid stream that polymerizes to yield the separating gel89. A second delivery tube 90 can be made to extend down inside theupper, wider section of the gel mold, and is used for the introductionof the second gel phase (the stacking gel 91) and (by means of switchinga valve) an equilibration solution used to bathe the IPG applied to theslab gel. A third delivery tube 95 also can be made to extend into theupper section of the gel mold, and is used to introduce the liquid thatpolymerizes into the top reservoir gel phase.

[0124] A slot form 92 can be lowered into the open top of the moldcavity by vertical movement of the slot form. The mold can be opened bymeans of another movement, whereby one face of the mold pivots along aline near to and parallel with the bottom horizontal edge of the moldcavity to expose the gel. The mold cavity contains indentations ateither end shaped so as to receive and support the ends of a carbonelectrode rod 94 and suspend it inside the top gel volume during itspolymerization. After polymerization of the gel, electrode rod 94 servesas both an upper electrode required for the electrophoresis separationand a mechanical support from which the gel hangs during subsequenthandling and manipulation. A further controlled motion is provided toclamp the electrode rod to one face of the gel mold, thus ensuring thatthe gel will always be recovered in a fixed location after the mold isopened.

[0125]FIG. 8 illustrates the sequence of actions of slab gel mold duringthe casting operation. In FIG. 8A, a slab gel mold comprised of a fixedmold half 86 and a movable mold half 87 is shown in the closed position.A long delivery tube 88 is extended downwards to the bottom of the mold,and the polymerizable mixture which will form the separating gel isdispensed. The motions of this tube and other delivery tubes arecontrolled by simple vertical electromechanical movements. In FIG. 9B,after the separating gel 89 is polymerized, a second shorter deliverytube 90 is lowered and a stacking gel phase is dispensed. In FIG. 9C,before the stacking gel 91 polymerizes, a slot form 92 is inserted intothe mold to form the sample slot 93. In FIG. 9D, once the stacking gelis polymerized, the slot form is withdrawn, an electrode rod 94 isinserted into the mold, and a third delivery tube 95 is lowered into themold to dispense a top gel mixture. In FIG. 9E, after the top gel 96 ispolymerized, the mold is opened. Once the mold is opened, a completedslab gel 97 hanging from the electrode rod 94 is slowly and evenlyremoved by slab gel handling arm 98 having an actuated gripper 99. Thearm is carried vertically and horizontally by linear motion components100 and 101.

[0126]FIGS. 9A through 9K illustrate alternative forms of slab gels. Thepreferred form of slab gel shown in FIG. 9A comprises three gel phases(separating gel 89, stacking gel 91, and top gel 96), an internalslot-shaped cavity 93 to accommodate the IPG first dimension gel 46, anda rod-shaped electrode 94. In FIG. 9B, the stacking gel phase iseliminated and the internal slot 93 is formed directly in the separatinggel 89. In FIG. 9C, the sample slot 93 extends to the top gel surface,while two internal electrode rods 94 a and 94 b are used. In FIG. 9D,the sample slot 93 also extends to the upper surface, but the electroderods 102 a and 102 b are external to the gel and support it byinteracting with lips 103 on the gel's external surfaces. In FIG. 9E,the IPG gel 46 is applied to an external face of the stacking gel phaserather than being placed in an internal slot, remaining in place as aresult of surface tension. In FIG. 9F, the IPG gel 46 is also appliedexternally, but to the separating gel 89 (the stacking gel 91 havingbeen eliminated). In FIG. 9G, the top phase 96 of a gel configured as inFIG. 9E is rotated counterclockwise by approximately 160 degrees. Byrotating the incorporated electrode rod 94, the top gel phase 96 isbrought in contact with the separating gel 89, bypassing the stackinggel 91 phase and the IPG gel 46, after sample proteins have entered intothe separating phase.

[0127] A series of alternative embodiments make use of a gel clamp,instead of a distinct gel region, to provide an electrode and source ofions. In FIG. 9H, a hinged clamp, comprised of halves 104 and 105,grasps the top edge of a slab gel and holds it as a result of theclosing force exerted by spring 106. One of the two opposing faces (105)contains an internal cavity 107 and electrode 108, the cavity forming aliquid-tight vessel when the gel is clamped in place thereby coveringopening 109. The gel is prevented from slipping out of the clamp by thepresence of a region of increased gel thickness 110 along the top geledge, in this case including a molded-in rod 111 as a means of handlingthe gel before introduction into the clamp, and secondarily by thepresence of a gritty coating on one or both of the opposing faces of theclamp. Projections 112 above the clamp's axis 113 can be squeezedtogether to open the clamp and release the gel. Axis 113 is connectedelectrically to the liquid vessel's electrode. An IPG gel 46 is appliedon the surface of the slab gel. Once the gel is grasped and the chamber107 is filled with an appropriate volume of electrode buffer, theassembly can be grasped in turn by external means via axis 113, andmanipulated by a robot arm as in the case of the gels with incorporatedelectrode rods (e.g., FIG. 9A). The electrode buffer solution providesthe source of ions for electrophoresis, using the axis 113 as aconvenient external electrical contact.

[0128] In FIG. 9I, a similar clamp is used to grasp a planar slab gelhaving no region of increased gel thickness along the top gel edge. Thegel is prevented from slipping out of the clamp only by the graspingforce and the presence of a gritty coating 114 on one or both opposingfaces of the clamp. In FIG. 9J, the IPG gel is placed within the clampon a support structure 115, and thereby held against the slab gel. Thebuffer-containing internal cavity is formed to provide two paths ofcurrent flow 116 and 117 into the slab gel: one above and a smaller onebelow the IPG. This arrangement provides a means for directing theproteins transported from a surface-applied IPG during electrophoresisinto the center plane of the slab gel. Hence, instead of moving alongthe surface of the slab to which they were applied (in the case wherethe IPG is applied to a surface, rather than inside of the slab), theprotein zone is pushed towards the interior of the gel by the flow ofbuffer ions entering through the second path 117. In FIG. 9K, the clampcontains a channel 118 through which buffer can be circulated. One legof this channel 118 runs along the top edge of the slab gel, where oneof the channel's walls is comprised of the gel's surface, and containsan electrode 108. This channel further communicates through additionalpassages 119 with an external buffer circulation system. In thisembodiment, buffer is circulated through the clamp during the run,providing a supply of fresh buffer components which, with the electrodemounted in the channel, allow sustained electrophoresis with a minimumvolume of reagents.

[0129] In the preferred embodiment, a separating gel (usually a gradientcomposition varying between approximately 18% T acrylamide at the bottomof the gel mold to 11% T acrylamide at the top of the separating gelphase) is introduced through the first delivery tube 88 (FIG. 8A) whileit is extended to the bottom of the mold cavity. This gradient isproduced by a second gradient maker similar in structure to thatdisclosed above to create an IPG gradient, except that larger syringesare used to produce a total separating gel volume of approximately 200ml. After the gel is introduced, the first delivery tube 88 is raisedout of the mold so that its open end lies in a block with vacuumchannels that direct a stream of air across the end of the tube and thusaspirate emerging liquid into a waste container. Multiport valvesassociated with the gradient maker syringes are switched so that thesyringes may be refilled, and so that a supply of pressurized water isconnected with the manifold leading to the delivery tube, thus purgingit of polymerizable components and flushing it with water. Thesetechniques for providing and aspirating delivery wash solvent functionin a manner similar to that described above for IPG gel formation. Theseparating gel is left undisturbed to polymerize for approximately 5minutes.

[0130] After initial polymerization, a second gel phase, a stacking gel91, is formed by extending the second delivery tube 90 into the top ofthe mold and dispensing approximately 50 ml of polymerizable stackinggel mixture directly atop the separating gel. The stacking gel 91 mix isformed by combining the output of three computer-controlled syringesdelivering stacking gel mix, ammonium persulfate and TEMED. Before thisgel phase polymerizes, the slot form 92 is caused to move down into thetop of the slab gel mold. The slot form 92 consists of a thin strip (˜1mm thick) of plastic mounted so as to present a vertical edge that lieson the mold center line which extends to within 1 cm of the separatinggel top and within 1 mm of the diverging walls of the mold in thejoining region. The slot form 92 is approximately 58 cm wide, leaving a1 cm open space at either of its ends.

[0131] The stacking gel 91 volume is so contrived that the joiningregion is filled with stacking gel mixture up to a depth on the slotform of approximately 3 mm. Upon polymerization of the stacking gel 91,the slot form 92 thus creates a slot 3 mm deep in the horn-shapedstacking gel cross-section, into which an IPG gel 46 or other proteincontaining sample may be placed.

[0132] After polymerization of the stacking gel 91, the slot form 92 iswithdrawn from the mold, and the arm system used for IPG manipulation isused to place an IPG strip in the slot so formed. Once this arm is againremoved from the mold area, the second delivery tube 90 is once againintroduced into the mold, and a volume of IPG equilibration solution isdispensed through it into the slot occupied by the IPG. Thisequilibration solution (consisting of 10% glycerol, 5 mM DTT, 2% SDS,0.125M Tris HCl pH 6.8 and a trace of bromophenol blue) serves to infuseSDS into the IPG gel 46 and alter its pH to that of the stacking gel 91in preparation for stacking. The second delivery tube 90 is then onceagain removed from the mold.

[0133] A second movable arm system, as shown in FIG. 12, then carries acarbon electrode rod 94 (or rods 99) to the mold and positions it withinthe mold, approximately 1 cm from the top of the mold cavity. Theelectrode rod ends rest in indentations at the ends of the mold cavity,maintaining the rod in position when released by the arm, which movesaway from the mold after depositing the rod. The third delivery tube 95is then introduced into the mold where it dispenses the third gel phase96 (the top gel), filling the mold to the top. This top gel phase 96 isproduced by a peristaltic pump system combining four components: anacrylamide/bis solution, a buffer solution, ammonium persulfate andTEMED.

[0134] The result is a slab gel in three phases, with the IPG firstdimension gel 46 and a carbon electrode rod 94 polymerized inside. Thepolymerizable gel solutions for these three phases are designed topolymerize rapidly, so that the three phases adhere to one another andyield an integral gel whose regions have distinct electrochemicalproperties.

[0135] Preferred compositions for the three phases are as follows.Acrylaide™ (FMC Corporation) is an alternative gel crosslinker which maybe used to increase gel strength in the stacking gel.

[0136] Separating gel

[0137] Acrylamide 13.00% T

[0138] bis acrylamide 3.8% C

[0139] Tris HCI pH 8.6 0.375M

[0140] Stacking gel

[0141] Acrylamide 8.00% T

[0142] Tris HCl pH 7.0 0.375M

[0143] Acrylaide 2% 3.2% C

[0144] SDS 0.2%

[0145] Top electrode gel

[0146] Acrylamide 13.00%T

[0147] Tris base 0.048M

[0148] Glycine 0.4M

[0149] SDS 0.20%

[0150] After the gel is made, the mold is opened by moving apart themold halves 86 and 87 and leaving the gel on the movable, now nearlyhorizontal, mold half 87. A second computer-controlled arm system,equipped with two graspers or pincers 99 designed to engage the oppositeends of the electrode rod 94, is moved into position to seize theelectrode rod 94 and then lift the gel upward and out of the mold.Gravity causes the gel to hang downwards from the bar.

[0151] Slab Gel Electrophoresis

[0152] The arm is then moved laterally into position over an empty slotin a slab gel running tank and slowly lowers the slab gel into the slot.FIG. 10 illustrates a slab gel running tank in accordance with thepresent invention, wherein a slab gel 97 is suspended vertically insilicone oil during the second dimension electrophoresis run. The slab97 is suspended by electrode rod 94 which rests on electrical bus bars120 (one at either end of the gel), with the slab gel 97 inserted into avertical slot through which cooled silicone oil is circulated. The oilcirculation path is so contrived as to cause laminar flow of a curtainof oil downwards along both surfaces of the slab gel, thereby removingjoule heat generated during electrophoresis. The oil is recovered at thebottom of the slots and recirculates through an external pump and heatexchanger, and thereafter is reintroduced into the top of the slot in aclosed-loop system. This curtain-like flow of oil serves to prevent theslab gel 97 from touching the walls of the slot, and insulates it fromelectrical contact along its length. Oil enters the tank throughmanifold 121, is distributed to supply plenums 122, expelled thoughholes 123 into the gel slot, and flows down the slot on either side ofthe separating gel 89, to be sucked out through return manifold 124 viareturn plenums 125 and return holes 126.

[0153] At the bottom of the tank, below the level of the bottom of theslots, a lower electrically-conductive aqueous phase 127 (denser thanthe silicone oil) is positioned so that it just contacts the bottom edgeof the slab gel 97. Current passes from the electrode bar or barsembedded in the top gel 96 through the stacking gel 91 and separatinggel 89 to the lower aqueous phase and lower electrode 128, thuscompleting the circuit required for an electrophoretic separation. Theshield 129 is provided over the lower electrode 128 to funnel thebubbles generated there to one side and up a separate pipe, thuspreventing their rising through the aqueous phase and then the siliconeoil phase, and causing mixing of the two phases.

[0154] At a voltage of 600 volts and a current of 1 amp, the separationof proteins in the separating gel 97 can be effected in approximately 4to 5 hours. Once the separation is complete, the aforementioned slab gelarm system is used to grasp the ends of the electrode bar 94, raise thegel out of the running slot and move the gel into position over thefirst of several tanks containing solutions required to visualize theseparated proteins by staining.

[0155] Slab gels and electrophoresis methods of the type disclosed canbe used for separation of samples other than proteins contained in IPGgels. In particular, the inclusion of multiple sample wells in place ofthe single slot provided for an IPG allows use of such gels to separateprotein or nucleic acid components of numerous liquid samples.

[0156] Slab Gel Staining

[0157] Several stain protocols can be executed including, among manyothers, staining with Coomassie Brilliant blue, ammoniacal silver,silver nitrate, and fluorescent stains such as SYPRO red and orange. Thefollowing example exemplifies the method applied to any stain. The gelis moved between subsequent tanks, by the arm under computer control, sothat the precise time of movement from one solution to the next can becontrolled, and can be held generally constant from gel to gel.

[0158] In a first tank, the gel is immersed up to the stacking gel in asolution of 30% ethanol, 2% phosphoric acid and 68% water for a periodof two hours, to fix the proteins in place and remove most of the SDS,Tris and glycine in the gel. Following this fixation step, the gel ismoved, through use of the arm, to a tank of 28% methanol, 14% ammoniumsulfate, 2% phosphoric acid in water, where it is incubated for twohours. Next, the gel is moved to a tank of the same composition with theaddition of powdered Coomassie Blue G250 dye, the whole liquid volumebeing continually circulated or agitated in the tank. Here the dyepermeates the gel, binding to resolved protein spots. Finally, the gelis removed from this tank and transported by the arm to a scanningstation.

[0159]FIG. 11A illustrates slab gel staining tanks with a slab carrierarm. In order to expose slab gels 97 to staining solutions, the gels aresuspended in staining tanks 130, where they are supported by theembedded electrode rods 94 whose ends sit on projecting supports 131.The tank 130 is filled with stain solution 132, which can be removedfrom the tank by opening exit valve 133. The tank 130 can be refilled byclosing valve 133 and then opening input valve 134 and activating pump135 to deliver solution 132 from reservoir 136. Solutions in the tankcan be agitated when required by a variety of means well known in thephotographic processing industry, including bursts of inert gas (such asnitrogen or argon) introduced at the bottom of the tank, or by smallmechanical motions of the suspended gels caused by cyclic movement ofthe gel supports 131. Gels 97 are moved from tank to tank by means ofarm 98 having pneumatically controlled grippers 99 which seize the endsof electrode rod 94. The arm 98 is raised and lowered by verticalmovement 100 which in turn rides on lateral movement 101, all undercomputer control.

[0160]FIGS. 11B and 11C show alternative embodiments allowing gelswithout incorporated electrode rods to be similarly processed. In FIG.11B, a slab gel 89 is contained inside a holder whose two halves 137 and138 are connected by hinge 139 at the top edge and held together bymagnets 140 at the bottom edge. Each half of the rectangular holder hasa large cutout and is shaped like a picture frame. One surface of eachhalf is covered with a taut mesh 141, resulting in a narrow gel cavitywith large-area porous walls. A slab gel placed in such a holder is thusexposed to any solution into which the holder is immersed, and can beprocessed through a series of tanks using a robot arm to graspprojecting pins 142. In FIG. 11C, an alternative slab gel holder makesuse of a clamp hinged at 142, held together by magnets 143 and havingits internal faces 144 coated with a gritty coating, to grasp a slab gelfor transportation and processing. Projections 145 may be squeezedtogether to open the clamp, releasing the gel.

[0161] Scanning

[0162] In order to obtain quantitative data on the abundance of resolvedproteins, the gel is scanned to yield a digitized image. FIG. 12 shows agel 97 being gently laid down on a horizontal or tilted illuminatingtable 146 prior to scanning, grasped as before by the electrode rod 94embedded in its top phase 96. To do this, the robotic arm 98 executes acoordinated vertical and horizontal motion so that the gel is laid downsmoothly without tension. An overhead digital camera 147, such as a CCDdigitizer, may then be used to acquire an image of the gel 97 and itsstained protein spots in absorbance mode. In order to allow scanning ofa large area gel at high resolution, a camera covering, for example,1024×1024 pixels can be moved to a series of locations by orthogonallinear motions 148 and 149, generating a series of scans that can becombined to yield a larger image. Alternative scanning and illuminationmodes may be provided for measuring fluorescence or light scattering, insituations where the proteins have been stained with a fluorescent or aparticulate dye, respectively. In the preferred embodiment, fluorescenceexcitation illumination is delivered to the gel in the plane of the gelwhile it lies in a horizontal cavity defined by walls 151 and filledwith a liquid 152, such as water, having a refractive index similar tothe gel. Light is piped into the cavity by an optical fiber light pipe153, one of whose ends pierces the walls 151, the other end beingilluminated by light produced by light source 155 filtered by interposedoptical filter 154. In fluorescence mode, light emitted by fluorescentmoieties in the gel is detected by the digitizer after passage through asecond optical filter 150 which passes the dye's emission wavelengthwhile blocking the excitation light. The approach described makes use ofthe fact that the exciting light is trapped by internal reflections inthe gel/water plane, thus improving its availability to exciteprotein-bound fluorescent dye molecules and diminishing the amount ofexciting light that escapes normal to the gel plane to impinge on thedetector. A similar optical system, but without a requirement forexcitation and emission filters, can be used to detect light scatteringby particles generated either on the protein spots (for example by thesilver stain) or around the spots (leaving the proteins negativelystained, as occurs with the copper stain).

[0163] Using the automated staining system described, multiple stain andscan cycles can be sequentially applied to the same gel. By stainingfirst with a relatively low sensitivity stain such as Coomassie Blue andscanning, and then staining with a relatively sensitive stain such asthe silver stain and scanning once again, it is possible to obtainquantitative protein abundance measurement over a wider dynamic rangethan can be afforded by any single conventional stain.

[0164] Multiple sequential scans of the same gel may be used to increasethe precision and dynamic range of non-equilibrium stains such as thesilver stain. In such stains, the development process reveals first theintensely staining spots (in general the more abundant proteins), thenthose of moderate staining intensity, and finally those of low stainingintensity (typically low abundance proteins), at which point theintensely staining spots are over stained, being saturated in stainabsorbance and appearing increased in size. By scanning the gel two ormore times during development, quantitation of spots can be based onmeasurements of parameters other than simple optical density. The mostuseful of such Parameters include maximum rate of change of absorbance(effectively the maximum slope observed in a plot of optical densityversus time) and time of onset of development (the time after thebeginning of development at which a given increment of optical densityis observed), both of which can be calculated for each pixel in thescanned gel image through use of multiple scans yielding optical density(or transmittance) as a function of time during the development of thegel. Alternatively, sophisticated curve-fitting algorithms can be usedto devise functions of absorbance as a function of time that yield, foreach pixel, a derived parameter well-correlated with known differencesin abundance.

[0165] Multiple scans of the same gel can also be used to compareprotein samples, provided that the proteins of each sample are labeledprior to electrophoresis with a dye or other substituent that can bedetected separately from other such labels. Multiple samples labeledwith a series of different fluorescent dyes having distinct emissionwavelengths, for example, can be mixed and co-electrophoresed. By usingappropriate optical filters to detect these dyes (and thus the proteinsto which they are bound) separately, the protein content of each samplecan be measured separately from the protein contents of other samplesapplied to the same gel. When used in a 2-D procedure that includesisoelectric focusing, such labels must be attached to the protein insuch a way that the protein's net pI is unaffected: if, for example, thelabel is attached by reaction with a lysine primary amino group, thenthe label must have a net charge of +1 to compensate for the singlepositive charge of the primary amino group lost when the lysine isderivatized. While this approach increases the information output ofeach separation (by multplexing samples), it also makes possible asubstantial increase in net resolution available for the comparison ofsamples. This comes about because the different label distributionsobserved in a small gel region (a protein spot in a 2-D electrophoresispattern) can be compared with great sensitivity by mathematicaltechniques to determine whether the shape and location of a spot in onelabel channel is precisely the same as the shape and location of a spotin another label channel (both labels being detected on the same gelwhere they reveal the proteins of two different samples). Spotpositional differences detectable by this approach (using for example acorrelation coefficient to determine whether the spot profiles in twochannels are the same or different) can be on the order of 0.1 mm, farless than the 0.5-2.0 mm position difference typically required tocharacterize protein spots as being different when two different gelsare compared, or when two samples are co-electrophoresed on one gel andstained with a single stain. When applied to both dimensions of a 2-Dprocedure, this method of comparing potentially co-electrophoresingproteins can result in an effective 100-fold increase in net gelresolution (the product of an approximate 10-fold resolution increase ineach dimension). Such an approach is of particular value in comparingvery different protein patterns (for example different tissues), whereit is likely that different proteins with similar 2-D gel positions maybe encountered.

[0166] Spot Excision

[0167] Protein spots can be excised from the gel under computer controlonce their positions are established by the aforementioned scanning.FIG. 13 shows a mechanical cutter comprised of a block 156 in whoselower part a thin-wall tube 157 is mounted vertically to act as aspot-cutting punch. The block and all its components are mounted on amovable, computer controlled X-Y frame, suspended just above anddo-planar with the gel, such that the cutter 157 can be positioned overany spot to be excised from the gel. A plunger 158 is arranged so as tomove vertically within the punch. The plunger extends through a hollowcavity 160 in the block and exits through a second hole by means ofchannel containing an O-ring seal 159. The plunger is moved verticallyby an actuator 161, and the block is moved vertically by a secondactuator 162 having less force, and thus capable of being overridden byactuation of the plunger actuator. The gel to be cut 97 lieshorizontally on a flat plat 163, which can be identical to the scanningplatform/lightbox 146. In operation, the cutter performs a series ofsteps as shown in the figure. In FIG. 13A, the block is positioned overthe spot to be cut. In FIG. 13B, the plunger actuator is pressed down,forcing the plunger to protrude through the cutting tube 157 into closeproximity with the gel surface and further forcing the block partiallydown through interaction of collar 164 on the plunger with the block. InFIG. 13C, actuator 162 is forced down, forcing the cutter through thegel and into contact with the supporting plate 163. In FIG. 13D, theplunger actuator 161 is pulled upwards, moving the block up byinteraction of collar 164 with the block and simultaneously generatingsuction in the cutter tube so as to ensure that the cut gel plug 165 islifted away from the gel by the upwards motion. In FIG. 13E, the cutterhas been repositioned over a collection vessel 166, and the plungerforced down to expel the gel plug into the vessel. In FIG. 13F, withboth actuators in the up position, a stream of wash liquid is introducedthrough hole 167 in the block 156 so as to expel any contaminatingparticulate gel material remaining in the punch into a waste receptacle168. Under computer control, the spot cutting mechanism can excisehundreds of spots from a single 2-D protein separation, depositing themin 96-well plates or other vessels for subsequent analysis by othermeans such as mass spectrometry. In the preferred embodiment, the spotcutter mechanism is incorporated into the gel scanning system, thusallowing the gel to be cut in an automated fashion immediately followingcomputer analysis of the gel image obtained from the scanner. SystemScheduling Algorithms

[0168] Operating as a continuous production line, the automated 2-D gelsystem described must allow flexible scheduling of each component actionin the multi-step process required to make and run each gel. If everygel were run using the same protocol, it would be possible to design acompletely synchronous scheduling system in which each action recurredat precisely defined intervals. However, such a system is inherentlyinflexible and would not allow running successive gels with differentparameters (e.g., different IPG pH gradient, focusing volt-hours, ortime in a stain solution). In addition, any temporary halt required insuch a synchronous system, due for example to an equipment breakdown,would cause variable and unforeseen consequences at different stages ofthe process.

[0169] Hence in the preferred embodiment, a non-synchronous schedulingalgorithm is used in which a series of steps is laid out for the firstsample to be run, and these are entered into a database of actionsrequired, each step associated with a relative or absolute time at whichit should be executed. Then a second series of steps is laid out for thesecond sample to be run, and these are entered into the databaseincluding a start delay calculated so as to prevent any action requiredfor the second gel from being interfered with by any action required forthe preceding (first) gel. Additional gels are added in order by thesame procedure, ensuring in each case that the actions required for agel do not interfere with those required for previously entered gels.Actions to be entered include casting an IPG gel, transporting an IPGfrom the caster to a wash slot, transporting an IPG from a wash to adrying slot, casting a slab gel, moving a slab gel from mold to runningslot, moving a slab gel from a running slot to a stain slot, etc.Database entries take account of the time required to execute suchactions, e.g., the time to move a gel from one station to another or toempty and refill a stain tank. The sequence of operations required toeffect the processing of a series of gels, including interleaving ofactions on different gels, is readily obtained by retrieving from thedatabase a series of steps sorted by time of scheduled execution. Makinguse of the ability of database software to sustain multiple independentqueries, different software modules controlling specific parts of thehardware system may retrieve a subset of actions (in scheduled timeorder) appropriate to them.

[0170] The automated system is then operated under the control of one ormore computer programs which function by examining the database ofscheduled actions, selecting from the database those actions appropriateto the hardware components being controlled by that program, andexecuting them at the time specified in the appropriate database record.Hence, a single IPG manipulation arm will be caused to transport IPGgels at different stages of the process between the required slots andstations, actions on different gels thus being interleaved in a flexiblemanner. Since each gel is separately scheduled at the outset, it canhave a different protocol or different parameters than the preceding orsucceeding gel, without limitation.

[0171] Data Reduction

[0172] Scanned images of 2D protein patterns are subjected to anautomated image analysis procedure using a batch process computersoftware (e.g., Kepler® software system). This software subtracts imagebackground, detects and quantitates spots, and matches spot patterns tomaster 2D patterns to establish spot identities. The final data for a2-D gel, a series of records describing position and abundance for eachspot, are then inserted as records in a computerized relationaldatabase.

[0173] Other Uses and Embodiments

[0174] The methods disclosed herein can be used for a series ofalternative analytical applications including the analysis of DNA andRNA, as well as peptides. Either the automated IPG or slab gel systemcan be used for high-throughput one-dimensional analyses of relevantbiomolecules as well as for 2-D.

[0175] It will be appreciated that the methods and structures of thepresent invention can be incorporated in the form of a variety ofembodiments, only a few of which are described herein. It will beapparent to the artisan that other embodiments exist that do not departfrom the spirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

LIST OF REFERENCES

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[0178] 3. U.S. Pat. No. 4,130,470, entitled “Method For Generating ApH-Function For Use In Electrophoresis”, issued to Rosengren, A. E.,Bjellqvist, B. and Gasparic, V. on December 1978.

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[0182] 7. Altiand, K. and Altland, A., Pouring reproducible gradients ingels under computer control: new devices for simultaneous delivery oftwo independent gradients, for more flexible slope and pH range ofimmobilized pH gradients. Clin. Chem. 30 (12 Pt 1):2098-2103, 1984.

[0183] 8. Disclosure Document No. 342751, Anderson, N. L., entitled“Vertical Method for Running IPG Gel Strips”, Nov. 12, 1993.

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[0186] 11. U.S. Pat. No. 4,169,036, issued to Anderson, Norman G. andAnderson, Norman L., entitled “System For Loading Slab-Gel Holders ForElectrophoresis Separation”, Sep. 25, 1979.

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[0191] 16. Disclosure Document No. 393753 (Anderson, N. L., Goodman,Jack, and Anderson, N. G., entitled “Gel Strips for ProteinSeparation”), Feb. 23, 1996.

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[0195] 20. Disclosure Document No. 346229 (Anderson, N. L., entitled“Method for Using Pre-Cast Thin Gels in Vertical Slab Gel Apparatus”),Jan. 19, 1994.

What is claimed is:
 1. A method for preparing a sample protein solutionfor electrophoresis analysis, said method comprises steps of: a)preparing a column, which comprises of a first end, a second end and asize exclusion material, by equilibrating said size exclusion materialwith a first solvent; b) introducing a reagent into said first end ofsaid column to create a reagent zone; c) introducing a protein sampleinto said first end of said column to create a sample zone, wherein saidsample contains a plurality of protein molecules; d) allowing saidprotein molecules to flow through said column, wherein said proteinmolecules flow ahead of said sample zone, pass through said reagentzone, and pass into said first solvent while in said column; and e)collecting said protein molecules from said second end of said column.2. The method according to claim 1, wherein said protein moleculescomprise a plurality of sulfhydryl groups which chemically react withsaid reagent.
 3. The method according to claim 2, wherein saidsulfhydryl groups covalently react with a zwitterionic or unchargedalkylating agent, preventing oxidation of said sulfhydryl groups, andpreserving an isoelectric point of each of said protein molecules. 4.The method of claim 2, wherein said sulfhydryl groups are reactedstoichiometrically with a negatively-charged alkylating reagent, therebyshifting the isoelectric points of basic proteins towards neutrality. 5.The method according to claim 1 wherein said protein molecules reactwith one or more fluorescent or optically absorbing dyes in said reagentzone, thereby said protein molecules are rendered detectable by anoptical means during and after subsequent separation of said proteinmolecules.
 6. The method according to claim 3, wherein said reagent isS+2-amino-5-iodacetamido-pentanoic acid, iodoacetamide, iodoacetic acid,N-ethyl maleimide or a combination thereof.
 7. An apparatus forpreparing a gel medium for separating a plurality of protein molecules,said apparatus comprising: a) a movable mold half which, when placednear a gel-binding material, forms a mold cavity; b) means for movingsaid movable mold half, c) means for transporting said gel-bindingmaterial; and d) means for dispensing a polymerizable gel mixture intosaid mold cavity.
 8. The apparatus of claim 7 wherein an O-ring isbetween said movable mold half and said gel-binding material and whereinsaid O-ring contacts both said movable mold half and said gel-bindingmaterial.
 9. The apparatus of claim 7 wherein said means for dispensingcomprises a means for dispensing a variable composition of saidpolymerizable gel mixture, wherein a gradient of said gel mixture isformed.
 10. The apparatus of claim 7 wherein said gel mixture chemicallybonds to said gel-binding material.
 11. The apparatus according to claim7, wherein said apparatus further comprises means for removing saidgel-binding material from said mold cavity.
 12. The apparatus accordingto claim 7, wherein said means for dispensing said polymerizable gelmixture comprises a precision gradient maker, wherein said gradientmaker comprises a plurality of syringes coupled to a plurality of valvesand said valves are coupled to a plurality of reservoirs.
 13. Theapparatus according to claim 12, wherein said precision gradient makerfurther comprises: a) a delivery tube comprising a first end and asecond end, wherein said first end is coupled to said plurality ofvalves; and b) said second end of said delivery tube is inserted intosaid mold cavity.
 14. The apparatus according to claim 12, wherein saidprecision gradient maker further comprises: a) a delivery tubecomprising a first end and a second end, wherein said first end iscoupled to said plurality of valves; and b) one of said valves coupledto said second end so that said delivery tube can be alternativelycoupled to a lowest point in said mold cavity or coupled to a wastecontainer.
 15. The apparatus according to claim 12, wherein each of saidreservoirs contains a solution selected from the group comprising: awash solution, one of a plurality of acrylamide monomer solutions, anammonium persulfate solution and a tetramethylethylene diamine solution.16. The apparatus according to claim 7 further comprising means forcontrolling temperature of said gel mixture within said mold cavity. 17.The apparatus according to claim 13 wherein said valves are coupled tosaid reservoirs such that when said valves are switched to allowrefilling of said syringes said delivery tube is washed with anon-polymerizable solvent.
 18. The apparatus according to claim 17,wherein said apparatus further comprises a means for aspirating asolution from said delivery tube.
 19. The apparatus according to claim13 wherein further comprising means for smoothly withdrawing saiddelivery tube during liquid delivery so that said second end of saiddelivery tube remains at or just above the rising meniscus of liquid insaid mold cavity.
 20. A method of preparing a gel medium for separatinga plurality of protein molecules, wherein said method comprises stepsof: a) attaching a gel-binding material to a longitudinal mold cavity;b) dispensing a variable composition of a polymerizable gel mixture intosaid mold cavity through a delivery tube having an open end, whereinsaid open end is inserted into said mold cavity, wherein said gelmixture comprises a gradient and wherein said gel mixture becomeschemically bonded to said backing material; c) withdrawing said deliverytube from said mold cavity during said dispensing whereby said open endof said delivery tube remains at or just above the rising meniscus ofsaid gel mixture in said mold cavity; and d) removing said gel-bindingmaterial and said gel mixture from said mold cavity.
 21. The methodaccording to claim 20, wherein said gel medium is prepared and used forthe analysis of a single sample, wherein said sample comprises of saidprotein molecules.
 22. The method according to claim 20, wherein saidgel median has a pH gradient along its length, rending said gel mediumsuitable for use as an IPG gel.
 23. The method according to claim 20,wherein said delivery tube is washed with a non-polymerizable solventcomposition thereby preventing polymerization of said gel mixture insaid delivery tube.
 24. The method according to claim 20, wherein saidgradient is dispensed as part of a sequence of segments comprising: a) afirst segment similar in volume to volume of said delivery tube, whereinsaid first segment is dispensed before insertion of said delivery tubeinto said mold cavity; b) a second segment comprising the remainder ofsaid gradient, wherein said segment is dispensed after insertion of saiddelivery tube into said mold; and c) a third segment having a volumesimilar to volume of said first segment.
 25. A method of preparing a gelmedium for separating a plurality of protein molecules, wherein saidmethod comprises steps of: a) attaching a backing material to alongitudinal mold cavity; b) dispensing a variable composition of apolymerizable gel mixture into said mold cavity, wherein said gelmixture comprising a gradient, said gel mixture chemically bonding tosaid backing material, and said mold cavity having a longitudinalportion with a nonrectangular cross-section; and c) removing saidbacking material and said gel mixture from said mold cavity.
 26. Themethod according to claim 25, wherein said gel medium is prepared andused for analyzing a single sample, wherein said sample comprises saidprotein molecules.
 27. A gel assembly for separating a plurality ofproteins wherein said gel assembly comprises: a) a gel comprising alongitudinal gradient of titratable gel monomers; b) a strip of backingmaterial to which said gel is chemically bonded, said backing materialhaving a greater width than said gel; and c) a longitudinal groove orcavity in which a liquid sample is held during use by capillary forces.28. A method of separating a plurality of proteins where said proteinsare in a liquid sample, said method comprising steps of: a) applyingsaid liquid sample onto a surface of a gel medium, wherein said gelmedium comprising a pH gradient and is attached to a backing material;b) applying a voltage across said gel medium to effect a separation ofsaid proteins; c) subjecting said gel medium to conditions wherein thevolume of said liquid sample is substantially decreased throughimbibition of said liquid sample into said gel medium or through loss ofwater in said liquid sample to an insulating gaseous or a liquidenvironment of said gel, such that said proteins are substantiallyincorporated into said gel.
 29. The method of claim 28 wherein saidsample is applied to a surface of said gel wherein said surfacecomprises a hole internal to said gel, a groove formed in said surfaceof said gel, or a groove comprising an included angle between said geland an area of said backing material extending outwardly from said gel.30. An apparatus for processing a gel medium for separating a pluralityof protein molecules wherein said gel medium is fixed to a backingmaterial, wherein said apparatus comprises: a) a plurality of stations;b) means for loading said gel medium with said protein molecules; c)means for reversibly grasping said backing material; and d) means fortransporting said gel medium to said stations.
 31. The apparatus ofclaim 30, wherein one or more of said stations comprise means forwashing, dehydrating and rehydrating said gel medium.
 32. The apparatusof claim 30, wherein one or more of said stations comprise means forapplication of a voltage longitudinally across said gel medium.
 33. Theapparatus according to claim 30 further comprising means for holdingsaid gel medium in place at each of said stations. 34, The apparatus ofclaim 30, wherein each of said stations comprises a plurality of slotsinto which a plurality of gel mediums may be inserted.
 35. The apparatusaccording to claim 32, wherein one or more of said stations ismaintained at different voltages.
 36. A gel medium for separating aplurality of protein molecules, wherein said gel medium comprises: a) afirst segment, wherein said first segment is planar and provides amedium for separating said protein molecules; and b) a second segment,wherein said second segment is of greater thickness than said firstsegment and comprises a buffer reservoir for supplying ions.
 37. The gelmedium according to claim 36, wherein a rigid electrode is embeddedwithin said second segment and wherein said electrode is used to apply avoltage across said gel.
 38. The gel medium according to claim 37,wherein said electrode forms a handle for transporting said gel medium.39. The gel medium according to claim 36, wherein an internal slot isformed in said second segment during molding, the floor of said slotbeing at or near a junction between said first segment and said secondsegment.
 40. The gel medium according to claim 36 wherein a shallowexternal slot is formed in said first segment during molding, said slotrunning parallel to and nearby a junction between said first segment andsaid second segment.
 41. A gel medium according to claim 36 furthercomprising a third segment, wherein said third segment is interposedbetween said first segment and said second segment, said third segmenthaving characteristics of a stacking gel, wherein said protein moleculesare stacked between a first low molecular weight ionic species and asecond low molecular weight ionic species prior to effecting aseparation of said protein molecules in said first segment.
 42. The gelmedium of claim 36 further comprising a third segment wherein said thirdsegment is interposed between said first segment and said secondsegment, and wherein said third segment comprises a compositiondifferent from the compositions of said first segment and said secondsegment and wherein said third segment has greater strength andelasticity than said first segment and said second segment.
 43. A gelmedium for separating a plurality of protein molecules, wherein said gelmedium comprises two regions of distinct geometry wherein a first regionis essentially planar and provides a medium for separating a pluralityof macromolecules and a second region is non-planar and provides a meansfor suspending said gel by an edge in liquid or gaseous surroundings.44. The gel medium according to claim 43, wherein part of said gelmedium is polymerized around a rigid support.
 45. The gel mediumaccording to claim 44, wherein said rigid support is used to transportor suspend said gel medium.
 46. The gel medium according to claim 43,wherein said second region is formed with projections or indentationswhich facilitate grasping of said second region when transporting orholding said gel medium.
 47. The gel medium according to claim 43further comprising a third region wherein said third region isinterposed between said first region and second region, and wherein saidthird region has a different composition than said first region and saidsecond region and said third region displays greater strength andelasticity than said first region and said second region.
 48. The gelmedium according to claim 43, wherein a rigid electrode is polymerizedwithin said second region, said electrode extends outside of said secondregion at one or more locations and said electrode is used to apply avoltage across said gel medium.
 49. A method of performingelectrophoreses in an acrylamide gel, said method comprises steps of: a)polymerizing said gel in a mold; b) removing said gel from said mold byreversible mechanical interaction with a region of said gel; and c)performing an electrophoretic separation in said gel.
 50. The methodaccording to claim 49 wherein said gel is supported by means of a rigidmechanical component, wherein said component is at least partiallyenclosed within said gel.
 51. The method according to claim 49 whereinsaid gel comprises a first segment in which macromolecular separationoccurs and a second segment comprising a first buffer reservoir.
 52. Themethod according to claim 51 wherein a sample is applied to said gel byinserting said sample into a slot inside said gel such that the floor ofsaid slot is at or near a junction between said first segment and saidsecond segment.
 53. The method according to claim 51 wherein a sample isapplied to an external surface of said gel nearby a junction betweensaid first segment and said second segment.
 54. The method according toclaim 51 wherein said second segment partially encloses a rigidelectrode.
 55. The method according to claim 54 wherein said firstsegment comprises a first end which contacts said second segment and asecond end which is distal from said second segment and wherein saidsecond end contacts a second buffer reservoir at an electrical voltagedifferent from a voltage applied to said rigid electrode.
 56. The methodaccording to claim 55 wherein said gel is at least partially suspendedin an insulating fluid.
 57. The method according to claim 56 whereinsaid insulating fluid is less dense than fluid comprising said secondbuffer reservoir.
 58. The method according to claim 57 wherein saidinsulating fluid is cooled.
 59. The method according to claim 58 whereinsaid insulating fluid is circulated over a surface of said gel.
 60. Themethod according to claim 51 wherein said gel further comprises a thirdsegment which is a stacking gel.
 61. The method according to claim 49wherein said gel comprises a plurality of projections or cavities or acombination thereof for mechanically supporting said gel.
 62. The methodaccording to claim 61 wherein said gel comprises a first segment inwhich macromolecular separation occurs and a second segment comprising afirst buffer reservoir, and wherein said first segment comprises a firstend which contacts said second segment and a second end which is distalfrom said second segment and wherein said second end contacts a secondbuffer reservoir at an electrical voltage different from a voltageapplied to said rigid electrode.
 63. The method according to claim 62wherein said gel is at least partially suspended in an insulating fluidduring electrophoresis.
 64. The method according to claim 63 whereinsaid insulating fluid is less dense than fluid comprising said secondreservoir buffer.
 65. The method according to claim 64 wherein saidinsulating fluid is cooled.
 66. The method according to claim 65 whereinsaid insulating fluid is circulated over the surface of said gel. 67.The method according to claim 49 wherein said gel is grasped by aplurality of gripping movable jaws, wherein one or more of said jawscomprises a cavity and an electrode, said cavity forming a liquid vesselbounded in part by said gel, such that when said vessel is filled withan appropriate buffer solution, electrical contact is establishedbetween said electrode and said gel.
 68. The method according to claim67, wherein an edge of said gel is distal from said liquid vessel, saidedge contacting a second buffer reservoir at an electrical voltagedifferent from a voltage applied to said electrode.
 69. The methodaccording to claim 68 wherein said get is at least partially suspendedin an insulating fluid.
 70. The method according to claim 67 whereineach of said gripping movable jaws has a first face and a second face,each of said first face and said second face being in frictional contactwith said gel.
 71. The method according to claim 70, wherein each ofsaid first face and said second face comprises a plurality of smallsharp grit particles.
 72. The method according to claim 49, wherein saidgel is grasped by a plurality of gripping movable jaws, wherein one ormore of said jaws comprises an internal liquid channel having anexternal input and an external output, said channel having at least onesegment exposed to the surface of said gel such that a liquidcirculating through said channel contacts said gel.
 73. The methodaccording to claim 72, wherein an edge of said gel is distal from saidliquid channel, said edge contacting a second buffer reservoir at anelectrical voltage different from a voltage applied to said electrode.74. The method according to claim 73 wherein said gel is at leastpartially suspended in an insulating fluid.
 75. The method according toclaim 72, wherein each of said gripping movable jaw has a first face anda second face, each of said first face and said second face being infrictional contact with said gel.
 76. The method according to claim 75,wherein each of said first face and said second face comprises aplurality of small sharp grit particles.
 77. The method of detectingmacromolecules in an electrophoreses gel which method comprises the stepof suspending said gel by an edge of said gel in a solution or asequence of solutions such that said macromolecules are rendereddetectable.
 78. The method according to claim 77, wherein said gel istransferred from one solution to another by a movable arm.
 79. Themethod according to claim 77, wherein said gel is suspended by a rigidmember which is at least partially embedded within said gel.
 80. Themethod according to claim 77, wherein said gel is suspended from anon-planar region of said gel.
 81. The method according to claim 77,wherein said gel is suspended by a plurality of gripping movable jaws.82. The method according to claim 81, wherein said jaws comprisesprings, magnets, electrical solenoids, pneumatic pistons or hydraulicpistons.
 83. The method according to claim 81, wherein each of said jawshas a first face and a second face, each of said first face and saidsecond face being in frictional contact with said gel.
 84. The methodaccording to claim 83, each of said first face and second face comprisesa plurality of small sharp grit particles.
 85. A method of detectingmacromolecules in an electrophoresis gel which method comprises the stepof placing said gel in a holder having an internal cavity withdimensions similar to said gel, and suspending said gel in a solution ora sequence of solutions such that said macromolecules are rendereddetectable.
 86. A method for scanning a stained gel medium, wherein saidmethod comprises the steps of: a) grasping an edge of said gel medium;b) transporting said gel medium to a scanning station by a mechanicalmotion means; and c) placing said gel medium in a space illuminated by alight source and within view of a light detector.
 87. The methodaccording to claim 86 further comprising the step of detecting lightabsorption, light scatter, fluorescence, luminescence, or fluorographicemission of said gel medium.
 88. The method according to claim 86,wherein said gel medium is scanned by a position-sensitive opticalsensor, photodiode array camera, CCD camera, moving laser beam, or amoving scanning head.
 89. The method according to claim 86, wherein saidmechanical motion means supports said gel by a rigid support embedded insaid gel.
 90. The method according to claim 86, wherein said mechanicalmotion means supports said gel by interaction with a non-planar regionof said gel.
 91. The method according to claim 86, wherein saidmechanical motion means grasps said edge of said gel by a plurality ofgripping movable jaws.
 92. A method of scanning a stainedelectrophoresis gel, said method comprises the steps of: a) immersingsaid gel in a thin planar cavity filled with a liquid having arefractive index similar to that of said gel; b) introducing anilluminating light into said cavity approximately in the plane of saidgel, wherein said illuminating light is substantially internallyreflected in said cavity and thereby prevented from exiting said cavitynormal to a plane of said gel; and c) positioning an optical scannersuch that said gel is viewed from outside of said cavity along a line ofsight normal to said gel.
 93. The method according to claim 92, whereinsaid cavity is a shallow horizontal depression filled with said liquid,wherein said liquid is aqueous.
 94. The method according to claim 92further comprising the step of positioning a light absorbing surface onan opposite side of said gel from said optical scanner, wherein said gelis stained with a fluorescent dye.
 95. The method according to claim 94,wherein said illuminating light comprises a spectrum enriched in anappropriate excitation wavelength of said dye and depleted of anappropriate emission wavelength of said dye, and said light impinges onsaid optical scanner after passage through a filter which preferentiallyabsorbs said excitation wavelength and transmits said appropriateemission wavelength of said dye.
 96. The method according to claim 92further comprising the steps of: a) positioning a light absorbingsurface on an opposite side of said gel from said scanner; and b)differentiating a plurality of stained features on said gel frombackground by a greater or a lesser scattering of said illuminatinglight in a direction of said scanner, wherein said gel is stained with aparticulate stain.
 97. A method of establishing relative quantitation ofproteins resolved in an electrophoresis gel, said method comprisingsteps of: a) staining said gel such that a plurality of opticalproperties of a plurality of protein-containing regions in said gel areprogressively changed during a period of time; b) optically scanningsaid gel two or more times during said period; c) measuring each of saidoptical properties as a function of time and recording a time sequencefor said optical properties; d) deriving a mathematical index from saidtime sequence; and e) calculating relative protein abundance of said gelor other useful property of said gel from said index.
 98. The methodaccording to claim 97, wherein said gel is stained using a silver-basedstain, a negative stain based on interaction of a detergent or aplurality of ions in said gel with a plurality of copper ions or aplurality of zinc ions in said gel, a Schiff stain for carbohydrates, orother kinetic stain, wherein a plurality of rates of diffusion of aplurality of reactants or a plurality of rates of chemical reactionsdetermine said period of time.
 99. The method according to claim 98,wherein said optical properties comprise transmittance, absorbance,fluorescence, luminescence, light scatter or refractive index as afunction of time.
 100. The method according to claim 98, wherein saidmathematical index comprises a maximum rate of change of said opticalproperties as a function of time, or an increment of time relative to afixed time at which a given change in said optical properties isdetected, or a combination of said rate of change and said incrementwith one or more of said optical properties.
 101. A method establishingrelative quantitation of proteins resolved in an electrophoresis gelwherein: a) staining said gel by two or more staining procedures toreveal said proteins; b) optically scanning said gel to detect aplurality of stain results after or during each of said stainingprocedure; and c) collectively interpreting said stain results by meansof a plurality of mathematical equations to determine a single measureof protein abundance.
 102. The method of comparing the properties of aplurality of proteins which may be present in a plurality of samples,wherein: a) each of said samples is reacted with a protein labelingreagent capable of being detected separately from labeling reagents usedto label other samples to be compared; b) said samples are combined andsaid proteins they contain are separated by one or more separationsprocesses; c) each of said labeling reagents is quantitatively measuredat a plurality of points along said separation to yield a proteinprofile for each sample; and d) said profiles are compared by amathematical technique to estimate the likelihood that apparentlyco-separating proteins in different samples are identical.
 103. Themethod of claim 102 wherein each sample to be combined is labeled with afluorescent dye having a different emission spectrum.
 104. The method ofclaim 102 wherein said mathematical technique compares said profiles bycorrelation or by means sensitive to peak width, shape or position. 105.The method of claim 102 wherein said likelihoods of protein identityprovide a means of detecting non-identity that is more sensitive thanthe resolving power of said separations process to resolve two proteinsin a single sample.
 106. A method of excising a plurality of smallregions of an electrophoresis gel comprising resolved proteins, saidmethod comprising steps of: a) staining said gel; b) scanning said gelto yield a digitized image; c) inputting said image into an imageprocessor for identifying a plurality of protein-containing regions insaid gel; d) excising said protein-containing regions from said gel bymeans of a computer-controlled movable punch device on the basis ofcoordinates supplied by said image processor, wherein said punch devicecomprises a tube-like cutter and a central piston within said cutter,said cutter and said piston being moved coaxial by a computer-controlledmeans to cut, retrieve and expel a plug of said gel; and e) depositingsaid protein-containing regions in a plurality of vessels.
 107. A systemfor preparing an electrophoresis gel, wherein said system comprises acomputer and a software, and said computer and software control aplurality of electromechanical means for preparing said gel and forremoving said gel from a mold.
 108. The system according to claim 107,wherein said system further loads a sample onto said gel, wherein saidsample comprises a plurality of components.
 109. The system according toclaim 108, wherein said system further effects an electrophoreticseparation of said components within said electrophoresis gel.
 110. Thesystem according to claim 109, wherein said system further detects saidcomponents after electrophoresis and staining of said gel.
 111. Thesystem according to claim 110, wherein said system performs a twodimensional protocol by preparing a first gel and a second gel, loadingsaid sample onto said first gel, effecting a first separation of saidcomponents in said first gel, transferring said first gel to said secondgel, effecting a second separation of said components in said secondgel, and detecting said components.
 112. A method for preparing anelectrophoresis gel, said method comprises steps of: casting said gel ina mold; and removing said gel from said mold by a plurality ofelectromechanical means, wherein each of said electromechanical means iscontrolled by a computer and a software.
 113. The method according toclaim 112, wherein said computer and said software further controlloading of a sample onto said gel, wherein said sample comprises aplurality of components.
 114. The method according to claim 113, whereinsaid computer and said software further control detection of saidcomponents within said gel.
 115. The method according to claim 114,wherein said computer and said software further control detection ofsaid components within said gel.
 116. The method according to claim 115,wherein said computer and said software control: 1) preparation of afirst gel and a second gel, 2) loading of said sample onto said firstgel, 3) a first separation of said components in said first gel, 4)transferring of said first gel to said second gel, 5) a secondseparation of said components in said second gel, and 6) detection ofsaid components within said gel.
 117. A method for separating aplurality of protein molecules in a plurality of gel mediums, saidmethod comprising steps of: a) grasping an edge of each of said gelmediums; b) transporting each of said gel mediums to a plurality ofstations wherein said stations comprise a plurality of environments; andc) inserting each of said gel mediums into said stations for separatingsaid protein molecules.
 118. The method according to claim 117, whereinsaid gel mediums are held by a plurality of grasping means respectively.119. The method according to claim 118, wherein said grasping meanscomprise one or more springs, magnets, electrical solenoids, pneumaticpistons, or hydraulic pistons.
 120. A programmable system for separatinga plurality of protein molecules in a plurality of gel mediumsrespectively, said system comprising: a) means for preparing said gelmediums; b) means for leading each of said gel mediums with said proteinmolecules; c) means for separating said protein molecules in each ofsaid gel mediums; d) means for staining or otherwise revealing proteinsin each of said gels; and e) means for scanning each of said gel mediumsto detect resolved macromolecules.
 121. The programmable systemaccording to claim 120 further comprising a plurality of stations. 122.The programmable system according to claim 121, wherein each of said gelmediums is assigned thereto a plurality of time intervals for each ofsaid stations respectively.
 123. The programmable system according toclaim 121, wherein each of said gel mediums is assigned thereto aplurality of parameters for separating said protein molecules in each ofsaid gel mediums.
 124. The programmable system according to claim 122further comprising means for scheduling said time intervals for each ofsaid stations respectively.
 125. The programmable system according toclaim 124, wherein said means for scheduling is asynchronous.
 126. Theprogrammable system according to claim 122 wherein said time intervalsassigned to at least one of said gel mediums is different from said timeintervals assigned to at least a second of said gel mediums.
 127. Theprogrammable system according to claim 123 further comprising means forcontrolling said plurality of parameters assigned to each of said gelmediums.
 128. The programmable system according to claim 123 whereinsaid parameters comprise volt-hours.
 129. The programmable systemaccording to claim 123 wherein said parameters comprise a pH gradient ofsaid gel mediums.
 130. The programmable system according to claim 123,wherein said parameters assigned to at least one of said gel mediums aredifferent from said parameters assigned to at least a second of said gelmediums.
 131. The programmable system according to claim 123 furthercomprising means for grasping each of said gel mediums at each of saidstations.
 132. The programmable system according to claim 123 furthercomprising means for transporting each of said gel mediums to saidstations.
 133. The programmable system according to claim 122 furthercomprising a database, wherein a first plurality of steps and a firstplurality of execution times based on said time intervals for a firstsample are entered into said database, and a second plurality of stepsand a second plurality of execution times based on said time intervalsfor a second sample are entered into said database, said secondplurality of steps including a start time delay calculated so as toprevent any action required for said second sample from interfering withany action required for said first sample, and said system retrievessaid first and second plurality of steps and said first and secondplurality of execution times from said databases and carries out firstand second plurality of steps in time order.
 134. An integrated systemfor two-dimensional electrophoresis, said system comprising: a) meansfor preparing an isoelectric focusing gel; b) means for loading saidisoelectric focusing gel with a plurality of protein molecules; c) meansfor applying a first voltage across said isoelectric focusing gel; d)means for preparing a slab electrophoresis gel; e) means for loadingsaid isoelectric focusing gel onto said slab electrophoresis gel; f)means for applying a second voltage across said slab electrophoresisgel; and g) means for staining said slab electrophoresis gel.
 135. Theintegrated system according to claim 134, wherein said system isprogrammable.
 136. The integrated system according to claim 135, whereinsaid system can operate on a repeated basis.
 137. The integrated systemaccording to claim 135, wherein said system comprises means for scanningsaid gel.
 138. The integrated system according to claim 137, whereinsaid system comprises computer software means for extracting estimatesof protein abundance and position from an image generated by said meansfor scanning said gel.
 139. The integrated system according to claim137, wherein said system comprises computer software means for insertingsaid estimates of protein abundance and position into a database.